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Sample records for cytomegalovirus-infected fibroblasts induces

  1. Dasatinib-induced hemorrhagic colitis complicated with cytomegalovirus infection

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    Aya Nakaya

    2017-12-01

    Full Text Available A 69-year-old man with chronic-phase chronic myeloid leukemia was initially treated with 100 mg dasatinib once a day. Despite a major molecular response within 9 months, he developed hemorrhagic colitis 32 months after starting dasatinib. Colonoscopy identified multiple hemorrhagic ulcers in the transverse colon. The pathological findings indicated cytomegalovirus infection. Dasatinib was stopped and he was started on ganciclovir. Three months later, colonoscopy confirmed the disappearance of the hemorrhagic ulcers. Dasatinib is a second-generation tyrosine kinase inhibitor used to treat chronic myeloid leukemia. As a multi-kinase inhibitor that acts on SRC-family kinases, its broader off-target kinase-inhibitory activity may account for the adverse events of dasatinib. Although gastrointestinal bleeding is common in patients taking dasatinib, the combination of cytomegalovirus infection and hemorrhagic colitis in the absence of systemic immunodeficiency is rare. Based on this case of dasatinibinduced hemorrhagic colitis with cytomegalovirus infection, we describe a possible mechanism and effective treatment.

  2. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

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    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  3. Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells

    DEFF Research Database (Denmark)

    Fornara, O; Bartek, J; Rahbar, A

    2016-01-01

    Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express...... human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary...... GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co...

  4. Pathogenesis of developmental anomalies of the central nervous system induced by congenital cytomegalovirus infection.

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    Kawasaki, Hideya; Kosugi, Isao; Meguro, Shiori; Iwashita, Toshihide

    2017-02-01

    In humans, the herpes virus family member cytomegalovirus (CMV) is the most prevalent mediator of intrauterine infection-induced congenital defect. Central nervous system (CNS) dysfunction is a distinguishing symptom of CMV infection, and characterized by ventriculoencephalitis and microglial nodular encephalitis. Reports on the initial distribution of CMV particles and its receptors on the blood brain barrier (BBB) are rare. Nevertheless, several factors are suggested to affect CMV etiology. Viral particle size is the primary factor in determining the pattern of CNS infections, followed by the expression of integrin β1 in endothelial cells, pericytes, meninges, choroid plexus, and neural stem progenitor cells (NSPCs), which are the primary targets of CMV infection. After initial infection, CMV disrupts BBB structural integrity to facilitate the spread of viral particles into parenchyma. Then, the initial meningitis and vasculitis eventually reaches NSPC-dense areas such as ventricular zone and subventricular zone, where viral infection inhibits NSPC proliferation and differentiation and results in neuronal cell loss. These cellular events clinically manifest as brain malformations such as a microcephaly. The purpose of this review is to clearly delineate the pathophysiological basis of congenital CNS anomalies caused by CMV. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  5. Cytomegalovirus infection in pregnancy.

    Science.gov (United States)

    Kagan, Karl Oliver; Hamprecht, Klaus

    2017-07-01

    Due to the severe risk of long-term sequelae, prenatal cytomegalovirus infection is of particular importance amongst intrauterine viral infections. This review summarizes the current knowledge about CMV infection in pregnancy. A search of the Medline and Embase database was done for articles about CMV infection in pregnany. We performed a detailed review of the literature in view of diagnosis, epidemiology and management of CMV infection in pregnancy. The maternal course of the infection is predominantly asymptomatic; the infection often remains unrecognized until the actual fetal manifestation. Typical ultrasound signs that should arouse suspicion of intrauterine CMV infection can be distinguished into CNS signs such as ventriculomegaly or microcephaly and extracerebral infection signs such as hepatosplenomegaly or hyperechogenic bowel. Current treatment strategies focus on hygienic measures to prevent a maternal CMV infection during pregnancy, on maternal application of hyperimmunoglobulines to avoid materno-fetal transmission in case of a maternal seroconversion, and on an antiviral therapy in case the materno-fetal transmission have occurred. CMV infection in pregnancy may result in a severe developmental disorder of the newborn. This should be taken into account in the treatment of affected and non-affected pregnant women.

  6. Mondini dysplasia and congenital cytomegalovirus infection.

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    Bauman, N M; Kirby-Keyser, L J; Dolan, K D; Wexler, D; Gantz, B J; McCabe, B F; Bale, J F

    1994-01-01

    We report a case of bilateral temporal bone anomalies in a child with symptomatic congenital cytomegalovirus infection and severe, bilateral sensorineural hearing loss identified at 3 months of age. High-resolution temporal bone computed tomography (HRCT) revealed bilateral findings of a short, malformed cochlea lacking an interscalar septum, a short and wide internal auditory canal, and an enlarged vestibular aqueduct, features diagnostic of bilateral Mondini dysplasia. To determine the importance of this observation, we completed HRCT in five additional children between 7 months and 9 years of age who had evidence of symptomatic congenital cytomegalovirus infection. One child with profound sensorineural hearing loss had severe bilateral temporal bone dysplasia with a small cochlea lacking an interscalar septum, an abnormal vestibule, and a large cochlear aqueduct. Of the remaining four children, hearing thresholds ranged from normal to profoundly decreased, but their HRCT scans were normal to visual inspection. When inner ear dimensions of these temporal bones were compared with norms established by Pappas and coworkers, however, seven of the eight ears had short cochleas and narrow lateral semicircular canals, and three ears had short or narrow vestibules. These results indicate that congenital cytomegalovirus infection may cause anomalies or growth disturbances of the temporal bone.

  7. Incidence and risk of primary cytomegalovirus infection among ...

    African Journals Online (AJOL)

    Background: Primary cytomegalovirus infection in pregnancy remains a leading cause of congenital hearing loss and mental retardation worldwide. Most women acquired CMV infection horizontally from their infected children or younger children who were cross- infected at school or day care facilities. Over 90% of infected ...

  8. Peptide inhibition of human cytomegalovirus infection

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    Morris Cindy A

    2011-02-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB, a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Results Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS, several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF were infected with the Towne-GFP strain of HCMV (0.5 MOI, preincubated with peptides at a range of concentrations (78 nm to 100 μM, and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100

  9. EBI3 regulates the NK cell response to mouse cytomegalovirus infection

    DEFF Research Database (Denmark)

    Jensen, Helle; Chen, Shih-Yu; Folkersen, Lasse Westergaard

    2017-01-01

    Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection. The induc......Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection....... The induction of EBI3 protein expression in mouse NK cells is a late activation event. Thus, early activation events of NK cells, such as IFNγ production and CD69 expression, were not affected in EBI3-deficient (Ebi3-/-) C57BL/6 (B6) mice during MCMV infection. Furthermore, comparable levels of early viral...... replication in spleen and liver were observed in MCMV-infected Ebi3-/- and wild-type (WT) B6 mice. Interestingly, the viral load in salivary glands and oral lavage was strongly decreased in the MCMV-infected Ebi3-/- B6 mice, suggesting that EBI3 plays a role in the establishment of MCMV latency. We detected...

  10. [Redox-potential of blood and consistence of energoproviding defence system in cytomegalovirus infection in pregnancy].

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    Dzhikiia, I V; Rizhvadze, M A; Dzhangidze, M A

    2006-05-01

    We have studied the relationship between the hypoxic change and mitochondrial redox-potential disturbances in the mechanism of pheto-placental insufficiency in pregnancy with cytomegalovirus infection (CMV), detected by the positive anti-CMV-IgG titer and more then 4-fold increase of low avid anti-CMV-IgG. It was shown, that chronic CMV infection induces production of active forms of oxygen, peroxidation of structures and concurrently damage of mitochondria with essential decrease of ATP level. Results of the study have shown the important diagnostic value of estimation of hypoxic-oxidative damage induced by CMV infection. The results also revealed important relationships between the activity of the CMV infection and intensity of mitochondrial damage. On the basis of our investigations we suggest the additional diagnostic test (the determination of citozol NADH dependent isocitratdehydrogenaze activity) to evaluate the depth of CMV induced metabolic disturbances.

  11. Symptomatic primary cytomegalovirus infection in a HIV-positive pregnant woman.

    LENUS (Irish Health Repository)

    Bergin, Sarah

    2014-12-01

    We describe a case of symptomatic primary Cytomegalovirus infection in a HIV-positive pregnant woman on antiretroviral treatment with a CD4 count >200 × 10(6)\\/l requiring intravenous ganciclovir. No adverse consequences from ganciclovir or evidence of congenital Cytomegalovirus infection were found.

  12. Intelligence and Academic Achievement With Asymptomatic Congenital Cytomegalovirus Infection.

    Science.gov (United States)

    Lopez, Adriana S; Lanzieri, Tatiana M; Claussen, Angelika H; Vinson, Sherry S; Turcich, Marie R; Iovino, Isabella R; Voigt, Robert G; Caviness, A Chantal; Miller, Jerry A; Williamson, W Daniel; Hales, Craig M; Bialek, Stephanie R; Demmler-Harrison, Gail

    2017-11-01

    To examine intelligence, language, and academic achievement through 18 years of age among children with congenital cytomegalovirus infection identified through hospital-based newborn screening who were asymptomatic at birth compared with uninfected infants. We used growth curve modeling to analyze trends in IQ (full-scale, verbal, and nonverbal intelligence), receptive and expressive vocabulary, and academic achievement in math and reading. Separate models were fit for each outcome, modeling the change in overall scores with increasing age for patients with normal hearing ( n = 78) or with sensorineural hearing loss (SNHL) diagnosed by 2 years of age ( n = 11) and controls ( n = 40). Patients with SNHL had full-scale intelligence and receptive vocabulary scores that were 7.0 and 13.1 points lower, respectively, compared with controls, but no significant differences were noted in these scores among patients with normal hearing and controls. No significant differences were noted in scores for verbal and nonverbal intelligence, expressive vocabulary, and academic achievement in math and reading among patients with normal hearing or with SNHL and controls. Infants with asymptomatic congenital cytomegalovirus infection identified through newborn screening with normal hearing by age 2 years do not appear to have differences in IQ, vocabulary or academic achievement scores during childhood, or adolescence compared with uninfected children. Copyright © 2017 by the American Academy of Pediatrics.

  13. Cytomegalovirus infection in inflammatory bowel disease is not associated with worsening of intestinal inflammatory activity.

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    Alexandre Medeiros do Carmo

    Full Text Available Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.Among the 400 eligible patients, 249 had Crohn's disease, and 151 had ulcerative colitis. In the group of Crohn's disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine.The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel

  14. Colonic stenosis post-necrotizing enterocolitis in term newborn with acquired cytomegalovirus infection.

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    Marseglia, L; Manti, S; D'Angelo, G; Lima, M; Impellizzeri, P; Romeo, C; Gitto, E

    2015-01-01

    Necrotizing enterocolitis is a gastrointestinal emergency typical of premature infants. Intestinal strictures infrequently complicate medical or surgical treatment of necrotizing enterocolitis. Postnatal cytomegalovirus infection with gastrointestinal linvolvement has occasionally been described in subjects with necrotizing enterocolitis. We report the case of a full term infant presenting necrotizing enterocolitis, acquired cytomegalovirus infection and post necrotizing enterocolitis colonic stricture.List of abbreviations: necrotizing enterocolitis = NEC,cytomegalovirus = CMV. Celsius.

  15. Maternal and fetal cytomegalovirus infection: diagnosis, management, and prevention [version 1; referees: 3 approved

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    Robert F. Pass

    2018-03-01

    Full Text Available Congenital cytomegalovirus infection is a major cause of central nervous system and sensory impairments that affect cognition, motor function, hearing, language development, vestibular function, and vision. Although the importance of congenital cytomegalovirus infection is readily evident, the vast majority of maternal and fetal infections are not identified, even in developed countries. Multiple studies of prenatal cytomegalovirus infections have produced a body of knowledge that can inform the clinical approach to suspected or proven maternal and fetal infection. Reliable diagnosis of cytomegalovirus infection during pregnancy and accurate diagnosis of fetal infection are a reality. Approaches to preventing the transmission of cytomegalovirus from mother to fetus and to the treatment of fetal infection are being studied. There is evidence that public health approaches based on hygiene can dramatically reduce the rate of primary maternal cytomegalovirus infections during pregnancy. This review will consider the epidemiology of congenital cytomegalovirus infection, the diagnosis and management of primary infection during pregnancy, and approaches to preventing maternal infection.

  16. Cytomegalovirus infection in NICU admitted neonates in Boushehr

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    Maryam Sanjideh

    2016-01-01

    Full Text Available Background: Cytomegalovirus is the most prevalent cause of congenital infections and the most important cause of congenital deafness. Which it's spread is about 0.64% of all birth which differ based on geolocation, race and socioeconomically situations. This proposal accomplished in the end of July until middle of February 2014 with the goal of studying Cytomegalovirus infection distribution among newborns who are hospitalized in Bushehr Shohadaye Khalij Fars hospital NICU. Material & Method: 80 urine samples were collected between July until February 2014 in NICU of Bushehr Khalij Fars hospitalized neonates. Samples were tested by PCR method on urine samples to find if they are infected by cytomegalovirus. Results: Mean age of neonates was 30.59±9.30 days. Only one newborn under 30 days had Cytomegalovirus and 11 cases older than 30 days had positive reaction. The relation between age and CMV seropositivity was statistically valid (p<0.05.this means only 1.2% of newborns are CMV and 55% are older than 1 month. Conclusion: The pattern of CMV seropositivity shows that most infections may be acquired from environment. According to low prevalence of congenital CMV infection, there is no need to introduce preventive methods and following present guidelines is enough.

  17. Clinical and morphological characteristics of malformations in infants with congenital cytomegalovirus infection and congenital toxoplasmosis

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    L. Yu. Barycheva

    2015-01-01

    Full Text Available The results of following up infants with intrauterine infections and malformations were retrospectively analyzed. Infants with malformations were diagnosed as having congenital cytomegalovirus infection and congenital toxoplasmosis in 127 and 69 cases, respectively. The aim of the study was to characterize malformations in infants with congenital cytomegalovirus and congenital Toxoplasma infections. The infants with malformations in congenital cytomegalovirus infection were found to have higher mortality rates (61,4% than those with congenital toxoplasmosis (34,8%. Postmortem analysis indicated that there was a predominance of embryopathies in infants with congenital cytomegalovirus infection and that of fetopathies in those with congenital toxoplasmosis. The dead infants with congenital cytomegalovirus infection had more commonly developed visceral defects, including heart diseases, pneumopathies, gastrointestinal and genitourinary abnormalities; fetopathies of the central nervous system and eye were prevalent in congenital toxoplasmosis. The surviving children with congenital toxoplasmosis were more frequently observed to have disabling CNS and ocular sequels as obstructive hydrocephalus, infantile cerebral palsy, complete or partial blindness, and cerebrasthenic disorders than those with congenital cytomegalovirus infection

  18. Neck stiffness in Guillaine-Barre syndrome subsequent to cytomegalovirus infection

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    İbrahim Etem Pişkin

    2011-03-01

    Full Text Available Guillain-Barre syndrome is an acute inflammatory demyelinating polyradiculoneuropathy that can be seen at any age. The classic symptoms such as flaccid paralysis and areflexia are not always predominant in children. In this study, we presented a 3-year-old girl with Guillain-Barre syndrome associated with cytomegalovirus infection who referred with showed atypical symptoms including neck stiffness.

  19. PP65 antigenemia in the diagnosis of cytomegalovirus infection in AIDS patients

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    RC Capela

    2012-01-01

    Full Text Available Cytomegalovirus causes significant morbidity and mortality in AIDS patients and those having undergone bone marrow or another transplant. PP65 antigenemia is based on detecting viral antigen in peripheral blood leukocytes through immunochemistry and by monitoring the infection in immunocompromised individuals. The present study aimed to set up this diagnostic technique in AIDS patients with active cytomegalovirus infection and verify its occurrence in the Botucatu region of São Paulo state, Brazil. Fifty patients, 35 men and 15 women aged from 24 to 69 years, were recruited from those attended at the Department of Tropical Diseases of Botucatu Medical School, UNESP, and divided into three groups according to CD4+ T lymphocyte counts and antiretroviral treatment. The control group comprised bone marrow transplant patients. Fourteen AIDS patients with low CD4+ cell counts tested positive for PP65 antigenemia, which could predict cytomegalovirus infection and indicate prophylactic treatment.

  20. Probable neuroimmunological link between Toxoplasma and cytomegalovirus infections and personality changes in the human host

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    Roubalová Kateřina

    2005-07-01

    Full Text Available Abstract Background Recently, a negative association between Toxoplasma-infection and novelty seeking was reported. The authors suggested that changes of personality trait were caused by manipulation activity of the parasite, aimed at increasing the probability of transmission of the parasite from an intermediate to a definitive host. They also suggested that low novelty seeking indicated an increased level of the neurotransmitter dopamine in the brain of infected subjects, a phenomenon already observed in experimentally infected rodents. However, the changes in personality can also be just a byproduct of any neurotropic infection. Moreover, the association between a personality trait and the toxoplasmosis can even be caused by an independent correlation of both the probability of Toxoplasma-infection and the personality trait with the third factor, namely with the size of living place of a subject. To test these two alternative hypotheses, we studied the influence of another neurotropic pathogen, the cytomegalovirus, on the personality of infected subjects, and reanalyzed the original data after the effect of the potential confounder, the size of living place, was controlled. Methods In the case-control study, 533 conscripts were tested for toxoplasmosis and presence of anti-cytomegalovirus antibodies and their novelty seeking was examined with Cloninger's TCI questionnaire. Possible association between the two infections and TCI dimensions was analyzed. Results The decrease of novelty seeking is associated also with cytomegalovirus infection. After the size of living place was controlled, the effect of toxoplasmosis on novelty seeking increased. Significant difference in novelty seeking was observed only in the largest city, Prague. Conclusion Toxoplasma and cytomegalovirus probably induce a decrease of novelty seeking. As the cytomegalovirus spreads in population by direct contact (not by predation as with Toxoplasma, the observed changes are

  1. Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

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    Robin Cloarec

    Full Text Available Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells.In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15 and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments.Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1.In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental

  2. Radiation-Induced Differentiation in Human Lung Fibroblast

    International Nuclear Information System (INIS)

    Park, Sa-Rah; Ahn, Ji-Yeon; Han, Young-Soo; Shim, Jie-Young; Yun, Yeon-Sook; Song, Jie-Young

    2007-01-01

    One of the most common tumors in many countries is lung cancer and patients with lung cancer may take radiotherapy. Although radiotherapy may have its own advantages, it can also induce serious problems such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of α-SMA and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-β), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are related to fibrosis. Among them TGF-β with Smad signaling is known to be the main stream and other signaling molecules such as MAPK, ERK and JNK (3) also participates in the process. In addition to those above factors, it is thought that more diverse and complicate mechanisms may involve in the radiationinduced fibrosis. Therefore, to investigate the underlying mechanisms in radiation induced fibrosis, first of all, we confirmed whether radiation induces trans differentiation in human normal lung fibroblasts. Here, we suggest that not only TGF-β but also radiation can induce trans differentiation in human lung fibroblast WI-38 and IMR-90

  3. Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts.

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    Sana Bahri

    Full Text Available Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA and rosmarinic acid (RA was reported to cure bleomycin-(BLM-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.

  4. Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

    International Nuclear Information System (INIS)

    Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

    2003-01-01

    Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

  5. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  6. Peripheral Blood Leukocytes and Serum Nested Polymerase Chain Reaction Are Complementary Methods for Monitoring Active Cytomegalovirus Infection in Transplant Patients

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    PD Andrade

    2013-01-01

    Full Text Available BACKGROUND: Human cytomegalovirus is an important cause of morbidity and mortality in immunocompromised patients. Qualitative polymerase chain reaction (PCR has proven to be a sensitive and effective technique in defining active cytomegalovirus infection, in addition to having low cost and being a useful test for situations in which there is no need for quantification. Real-time PCR has the advantage of quantification; however, the high cost of this methodology makes it impractical for routine use.

  7. [Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].

    Science.gov (United States)

    Zhou, Yu-feng; Fang, Feng; Dong, Yong-sui; Zhou, Hua; Zhen, Hong; Liu, Jin; Li, Ge

    2006-07-01

    Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased

  8. Citomegalovirose congênita: relato de caso Congenital cytomegalovirus infection: a case report

    Directory of Open Access Journals (Sweden)

    Patrícia de Fátima Azevedo

    2005-12-01

    Full Text Available A citomegalovirose congênita sintomática é entidade clínica de grande importância devido a sua vasta sintomatologia fetal. No Brasil, o diagnóstico intra-útero é ainda pouco realizado, apesar do grande arsenal propedêutico. Relatamos um caso de citomegalovirose congênita grave com hepatoesplenomegalia, agenesia parcial do vérmix cerebelar, calcificações intracranianas, placentomegalia, aumento da ecogenicidade intestinal e renal, cardiomegalia, hipoplasia pulmonar, derrame pericárdico e ascite. A ressonância nuclear magnética fetal foi utilizada para confirmação dos achados ultra-sonográficos. A amniocentese foi realizada para análise do líquido amniótico por meio da PCR, sendo evidenciado resultado positivo. O óbito fetal foi constatado na 31ª semana de gestação, sendo confirmados os achados através da citopatologia e estudo anatomopatológico do natimorto. O arsenal propedêutico existente, na atualidade, para diagnóstico intra-útero da citomegalovirose congênita é de grande importância para confirmação diagnóstica e determinação do prognóstico fetal.Congenital cytomegalovirus infection is an important clinical entity, due to its sonographic symptomatology. In Brazil, in utero diagnosis is not accomplished despite the improvements in diagnostic methods. We report a congenital infection including: splenomegaly and hepatomegaly, hypoplasia of the cerebellar vermis, intracranial calcifications, hyperechoic kidneys, hyperechoic bowel, cardiomegaly, lung hypoplasia, ascites, and pericardial effusion. Fetal magnetic resonance imaging confirmed the sonographic findings. Amniocentesis was performed for cytomegalovirus PCR in amniotic fluid, which confirmed fetal infection. Fetal loss occurred in the 31st week of pregnancy. Necropsy studies confirmed the sonographic findings. The diagnostic methods have been useful to confirm congenital cytomegalovirus infection and to establish fetal outcome.

  9. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals......-treated fibroblasts. Transcriptional differences in basal and radiation-induced gene expression profiles were investigated using 15K cDNA microarrays, and results analyzed by both SAM and PAM. RESULTS: Sixty differentially expressed genes were identified by applying SAM on 10 patients with the highest risk of RIF...

  10. Hygiene interventions for prevention of cytomegalovirus infection among childbearing women: systematic review.

    Science.gov (United States)

    Harvey, Jessica; Dennis, Cindy-Lee

    2008-09-01

    This paper is a report of a systematic review to examine the effectiveness of preventive interventions to reduce congenital cytomegalovirus transmission and infection among women of childbearing age. Congenital cytomegalovirus has been identified as the leading infectious cause of damage to the growing fetus in developed countries, including Down syndrome, fetal alcohol syndrome and spina bifida. Despite the prevalence and consequences of this infection, it has a low profile and pregnant mothers are often unaware of the risks and protective behaviours related to its transmission. Women with children in daycare and nurses working with children are particularly at risk of acquiring the virus. A computerized literature search for articles up to 1 December 2007 was performed using MEDLINE (from 1950); EMBASE (from 1980) and CINAHL (from 1982). Both authors independently reviewed studies that met inclusion criteria and assigned a quality rating determined by the number of validity criteria met. Differences were discussed until consensus was reached. Differences in hygiene behaviour changes were most statistically significant for pregnant, seronegative women. Although the methodological quality of the three included studies was not strong, seroconversion rates consistently decreased as cytomegalovirus education and support increased. Nurses can act as preventive agents for cytomegalovirus infection through education about hygiene precautions during antenatal care and through preventive measures in the workplace. The review findings suggest educational interventions in hygiene practices have the potential to be a feasible, large-scale, primary prevention strategy.

  11. Congenital cytomegalovirus infection in pregnancy and the neonate: consensus recommendations for prevention, diagnosis, and therapy.

    Science.gov (United States)

    Rawlinson, William D; Boppana, Suresh B; Fowler, Karen B; Kimberlin, David W; Lazzarotto, Tiziana; Alain, Sophie; Daly, Kate; Doutré, Sara; Gibson, Laura; Giles, Michelle L; Greenlee, Janelle; Hamilton, Stuart T; Harrison, Gail J; Hui, Lisa; Jones, Cheryl A; Palasanthiran, Pamela; Schleiss, Mark R; Shand, Antonia W; van Zuylen, Wendy J

    2017-06-01

    Congenital cytomegalovirus is the most frequent, yet under-recognised, infectious cause of newborn malformation in developed countries. Despite its clinical and public health importance, questions remain regarding the best diagnostic methods for identifying maternal and neonatal infection, and regarding optimal prevention and therapeutic strategies for infected mothers and neonates. The absence of guidelines impairs global efforts to decrease the effect of congenital cytomegalovirus. Data in the literature suggest that congenital cytomegalovirus infection remains a research priority, but data are yet to be translated into clinical practice. An informal International Congenital Cytomegalovirus Recommendations Group was convened in 2015 to address these questions and to provide recommendations for prevention, diagnosis, and treatment. On the basis of consensus discussions and a review of the literature, we do not support universal screening of mothers and the routine use of cytomegalovirus immunoglobulin for prophylaxis or treatment of infected mothers. However, treatment guidelines for infected neonates were recommended. Consideration must be given to universal neonatal screening for cytomegalovirus to facilitate early detection and intervention for sensorineural hearing loss and developmental delay, where appropriate. The group agreed that education and prevention strategies for mothers were beneficial, and that recommendations will need continual updating as further data become available. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Oxidized DNA induces an adaptive response in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2013-07-15

    Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

  13. ROS Mediates Radiation-Induced Differentiation in Human Lung Fibroblast

    International Nuclear Information System (INIS)

    Park, Sa Rah; Ahn, Ji Yeon; Kim, Mi Hyeung; Lim, Min Jin; Yun, Yeon Sook; Song, Jie Young

    2009-01-01

    One of the most common tumors worldwide is lung cancer and the number of patients with lung cancer received radiotherapy is increasing rapidly. Although radiotherapy may have lots of advantages, it can also induce serious adverse effects such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of smooth muscle actin-alpha (a-SMA) and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-b), tumor necrosis factor (TNF), IL-6, platelet-derived growth factor (PDGF) and reactive oxygen species are related to fibrosis. It is also reported that reactive oxygen species (ROS) can be induced by radiation and can act as a second messenger in various signaling pathways. Therefore we focused on the role of ROS in radiation induced fibrosis. Here, we suggest that irradiation generate ROS mainly through NOX4, result in differentiation of lung fibroblast into myofibroblast

  14. Dioxin induces genomic instability in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Merja Korkalainen

    Full Text Available Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI, i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD. Mouse embryonic fibroblasts (C3H10T1/2 were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days. For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay, was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

  15. TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

    International Nuclear Information System (INIS)

    Chen, Xiao-Qing; Zhang, Dao-Liang; Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang; Jiang, Wei-Feng; Zhou, Li; Zhao, Liang; Wang, Quan-Xing; Liu, Xu

    2015-01-01

    Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage

  16. TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiao-Qing; Zhang, Dao-Liang [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-Feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhao, Liang, E-mail: zhaol_zg@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Wang, Quan-Xing, E-mail: wqxejd@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Liu, Xu, E-mail: liuxu_xk@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China)

    2015-08-14

    Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage.

  17. Mesenchymal stem cells induce dermal fibroblast responses to injury

    International Nuclear Information System (INIS)

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  18. Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke-induced alterations

    Directory of Open Access Journals (Sweden)

    James Elliot Scott

    2016-03-01

    Full Text Available Background Cigarette smoking is the leading cause of preventable death in the world. It has been implicated in the pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is clearly a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity is the first site of exposure to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases, are proteases which belong to the family of cysteine aspartic acid proteases and are the key components for the downstream amplification of intra-cellular apoptotic signals. Of the 14 caspases known, caspase-3 is the key executioner of apoptosis. Fetal rat lung fibroblasts but not PDL viability is reduced by exposure to CSE. In addition Caspase 3 activity is elevated after CSE exposure in fetal lung fibroblasts but not in PDLs. Expression of caspase 3 is induced in CSE exposed lung fibroblasts but not in PDLs. Caspase 3 was localized to the cytoplasm in both cell types.

  19. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    International Nuclear Information System (INIS)

    Nishiyama, Y.; Rapp, F.

    1981-01-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

  20. Evodiamine attenuates TGF-β1-induced fibroblast activation and endothelial to mesenchymal transition.

    Science.gov (United States)

    Wu, Qing-Qing; Xiao, Yang; Jiang, Xiao-Han; Yuan, Yuan; Yang, Zheng; Chang, Wei; Bian, Zhou-Yan; Tang, Qi-Zhu

    2017-06-01

    The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

  1. Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jon M Carthy

    Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

  2. SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

    Science.gov (United States)

    Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

    2018-05-01

    SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

  3. Human cytomegalovirus infection dysregulates the canonical Wnt/β-catenin signaling pathway.

    Directory of Open Access Journals (Sweden)

    Magdalena Angelova

    Full Text Available Human Cytomegalovirus (HCMV is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/β-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/β-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of β-catenin and expression of β-catenin target genes in these cells. HCMV infection leads to β-catenin protein accumulation in a discrete juxtanuclear region. Levels of β-catenin in membrane-associated and cytosolic pools, as well as nuclear β-catenin, are reduced after infection; while transcription of the β-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/β-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

  4. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  5. Normal formation and repair of γ-radiation-induced single and double strand DNA breaks in Down syndrome fibroblasts

    International Nuclear Information System (INIS)

    Steiner, M.E.; Woods, W.G.

    1982-01-01

    Fibroblasts from patients with Down syndrome (Trisomy 21) were examined for repair capability of γ-radiation-induced single strand and double strand DNA breaks. Formation and repair of DNA breaks were determined by DNA alkaline and non-denaturing elution techniques. Down syndrome fibroblasts were found to repair single strand and double strand breaks as well as fibroblasts from normal controls. (orig.)

  6. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    2011-02-01

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  7. Life-threatening intracranial bleeding in a newborn with congenital cytomegalovirus infection: late-onset neonatal hemorrhagic disease.

    Science.gov (United States)

    Dallar, Yildiz; Tiras, Ulku; Catakli, Tulin; Gulal, Gonul; Sayar, Yavuz; Selvar, Beray; Alioglu, Bulent

    2011-02-01

    The authors present a case of a 36-day-old infant with intracranial and intramuscular hemorrhage due to vitamin K deficiency bleeding, who received intramuscular vitamin K prophylaxis at birth. In this case, laboratory tests showed anemia, liver dysfunction with cholestasis, and coagulopathy, consistent with vitamin K deficiency abnormality. Serological analyses showed that cytomegalovirus immunoglobulin (Ig)M and IgG avidity were both positive. The infant was treated successfully with intravenous ganciclovir and blood products. This case suggests that it is imperative to meticulously investigate the etiology in neonates with late-onset hemorrhagic disease of the newborn. Cholestatic liver disease caused by congenital cytomegalovirus infection should be in mind in term infants who presented with late-onset hemorrhagic disease.

  8. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  9. Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts

    International Nuclear Information System (INIS)

    Copaja, Miguel; Venegas, Daniel; Aranguiz, Pablo; Canales, Jimena; Vivar, Raul; Catalan, Mabel; Olmedo, Ivonne; Rodriguez, Andrea E.; Chiong, Mario; Leyton, Lisette; Lavandero, Sergio; Diaz-Araya, Guillermo

    2011-01-01

    Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types. Methods: Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1-10 μM) up to 72 h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively. Results: Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras. Conclusion: Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues. - Research Highlights: → Simvastatin decreases CF and CMF viability independent of cholesterol synthesis. → Simvastatin induces CF and CMF apoptosis in a caspase-dependent manner being CMF more resistant

  10. An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    David T. Vereide

    2014-12-01

    Full Text Available During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that “trap” murine cells in a proliferative state and endow them with a hemangioblast potential. These “expandable” hemangioblasts (eHBs are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.

  11. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts

    International Nuclear Information System (INIS)

    Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J.

    2014-01-01

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction

  12. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, D.X.; Deng, T.Z.; Lv, J.; Ke, J. [Department of Stomatology, Air Force General Hospital PLA, Haidian District, Beijing (China)

    2014-09-19

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  13. Cell death induced by hydroxyapatite on L929 fibroblast cells.

    Science.gov (United States)

    Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

    2004-05-01

    Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

  14. Proteome oxidative carbonylation during oxidative stress-induced premature senescence of WI-38 human fibroblasts

    DEFF Research Database (Denmark)

    Le Boulch, Marine; Ahmed, Emad K; Rogowska-Wrzesinska, Adelina

    2018-01-01

    Accumulation of oxidatively damaged proteins is a hallmark of cellular and organismal ageing, and is also a phenotypic feature shared by both replicative senescence and stress-induced premature senescence of human fibroblasts. Moreover, proteins that are building up as oxidized (i.e. the "Oxi-pro...

  15. Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia.

    NARCIS (Netherlands)

    Imel, E.A.; Peacock, M.; Pitukcheewanont, P.; Heller, H.J.; Ward, LM; Shulman, D.; Kassem, M.; Rackoff, P.; Zimering, M.; Dalkin, A.; Drobny, E.; Colussi, G.; Shaker, J.L.; Hoogendoorn, E.H.; Hui, S.L.; Econs, M.J.

    2006-01-01

    CONTEXT: Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in

  16. Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia

    DEFF Research Database (Denmark)

    Imel, Erik A; Peacock, Munro; Pitukcheewanont, Pisit

    2006-01-01

    Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tumors t...

  17. Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts.

    Science.gov (United States)

    Hemmings, D G; Kilani, R; Nykiforuk, C; Preiksaitis, J; Guilbert, L J

    1998-06-01

    Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.

  18. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    International Nuclear Information System (INIS)

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S.

    2005-01-01

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCα-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis

  19. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

    Directory of Open Access Journals (Sweden)

    Rong Yao

    Full Text Available Pulmonary fibrosis is one of the most common complications of paraquat (PQ poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR. Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR 1 small-interfering RNA (siRNA group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8 and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05. Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05. APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a

  20. Replication-defective lymphocytic choriomeningitis virus vectors expressing guinea pig cytomegalovirus gB and pp65 homologs are protective against congenital guinea pig cytomegalovirus infection.

    Science.gov (United States)

    Cardin, Rhonda D; Bravo, Fernando J; Pullum, Derek A; Orlinger, Klaus; Watson, Elizabeth M; Aspoeck, Andreas; Fuhrmann, Gerhard; Guirakhoo, Farshad; Monath, Thomas; Bernstein, David I

    2016-04-12

    Congenital cytomegalovirus infection can be life-threatening and often results in significant developmental deficits and/or hearing loss. Thus, there is a critical need for an effective anti-CMV vaccine. To determine the efficacy of replication-defective lymphocytic choriomeningitis virus (rLCMV) vectors expressing the guinea pig CMV (GPCMV) antigens, gB and pp65, in the guinea pig model of congenital CMV infection. Female Hartley strain guinea pigs were divided into three groups: Buffer control group (n = 9), rLCMV-gB group (n = 11), and rLCMV-pp65 (n = 11). The vaccines were administered three times IM at 1.54 × 10(6)FFU per dose at 21-day intervals. At two weeks after vaccination, the female guinea pigs underwent breeding. Pregnant guinea pigs were challenged SQ at ∼ 45-55 days of gestation with 1 × 10(5)PFU of GPCMV. Viremia in the dams, pup survival, weights of pups at delivery, and viral load in both dam and pup tissues were determined. Pup survival was significantly increased in the LCMV-gB vaccine group. There was 23% pup mortality in the gB vaccine group (p = 0.044) and 26% pup mortality in the pp65 vaccine group (p = 0.054) compared to 49% control pup mortality. The gB vaccine induced high levels of gB binding and detectable neutralizing antibodies, reduced dam viremia, and significantly reduced viral load in dam tissues compared to control dams (p < 0.03). Reduced viral load and transmission in pups born to gB-vaccinated dams was observed compared to pups from pp65-vaccinated or control dams. The rLCMV-gB vaccine significantly improved pup survival and also increased pup weights and gestation time. The gB vaccine was also more effective at decreasing viral load in dams and pups and limiting congenital transmission. Thus, rLCMV vectors that express CMV antigens may be an effective vaccine strategy for congenital CMV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Adventitial fibroblasts induce a distinct proinflammatory/profibrotic macrophage phenotype in pulmonary hypertension.

    Science.gov (United States)

    El Kasmi, Karim C; Pugliese, Steven C; Riddle, Suzette R; Poth, Jens M; Anderson, Aimee L; Frid, Maria G; Li, Min; Pullamsetti, Soni S; Savai, Rajkumar; Nagel, Maria A; Fini, Mehdi A; Graham, Brian B; Tuder, Rubin M; Friedman, Jacob E; Eltzschig, Holger K; Sokol, Ronald J; Stenmark, Kurt R

    2014-07-15

    Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling. Copyright © 2014 by The American Association of Immunologists

  2. Adventitial Fibroblasts induce a distinct Pro-inflammatory/Pro-fibrotic Macrophage Phenotype in Pulmonary Hypertension

    Science.gov (United States)

    El Kasmi, Karim C.; Pugliese, Steven C.; Riddle, Suzette R.; Poth, Jens M.; Anderson, Aimee L.; Frid, Maria G.; Li, Min; Pullamsetti, Soni S.; Savai, Rajkumar; Nagel, Maria A.; Fini, Mehdi A.; Graham, Brian B.; Tuder, Rubin M.; Friedman, Jacob E.; Eltzschig, Holger K.; Sokol, Ronald J.; Stenmark, Kurt R.

    2014-01-01

    Macrophage accumulation is not only a characteristic hallmark but also a critical component of pulmonary artery (PA) remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Utilizing multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, as well as primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive Pas (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL4/IL13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation while complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, while deficiency in C/EBPβ or HIF1 attenuated fibroblast driven macrophage activation. These findings challenge the current paradigm of IL4/IL13-STAT6 mediated alternative macrophage activation as the sole driver of vascular remodeling in PH and uncover a crosstalk between adventitial fibroblasts and macrophages in which paracrine IL6 activated STAT3, HIF1, and C/EBPβ signaling is critical for macrophage activation and polarization. Thus, targeting IL6 signaling in macrophages by completely inhibiting C/EBPβ, HIF1a or partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL6 and absent IL4/IL13 signaling. PMID:24928992

  3. Typical xeroderma pigmentosum complementation group A fibroblasts have detectable ultraviolet light-induced unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Petinga, R.A.; Andrews, A.D.; Robbins, J.H.; Tarone, R.E.

    1977-01-01

    Ultraviolet-induced nuclear uptake of tritiated thymidine [ 3 H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported that the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [ 3 H]dThd at 37degC, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain count was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain count was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain count average of the irradiated cells was always higher than that of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of that given by normal non-xeroderma pigmentosum fibroblasts and was not reduced by 10 -2 M hydroxyurea. Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is not due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains

  4. Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts.

    Science.gov (United States)

    Chiu, Po Yee; Lam, Philip Y; Yan, Chung Wai; Ko, Kam Ming

    2011-06-01

    The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. S100A4 amplifies TGF-β-induced fibroblast activation in systemic sclerosis

    DEFF Research Database (Denmark)

    Tomcik, Michal; Palumbo-Zerr, Katrin; Zerr, Pawel

    2015-01-01

    (SSc). METHODS: The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4...... or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4(-/-) mice were protected from bleomycin-induced skin fibrosis with reduced dermal...

  6. Membrane damage induced in cultured human skin fibroblasts by UVA irradiation

    International Nuclear Information System (INIS)

    Gaboriau, F.; Morliere, P.; Marquis, I.; Moysan, A.; Geze, M.; Dubertret, L.

    1993-01-01

    Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells. (Author)

  7. Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

    Science.gov (United States)

    Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E.; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P.

    2013-01-01

    Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results

  8. Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

    DEFF Research Database (Denmark)

    Fusi, Elenora; Rebucci, Raffaella; Pecorini, Chiara

    2010-01-01

    The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity....... Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P tocopherol...

  9. PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

    Directory of Open Access Journals (Sweden)

    Mao-lin HUANG

    2014-09-01

    Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

  10. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    Science.gov (United States)

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  11. Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Zamansky, G B

    1986-08-01

    The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.

  12. Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

    International Nuclear Information System (INIS)

    Zamansky, G.B.

    1986-01-01

    The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells. (author)

  13. CLINICAL AND IMMUNOLOGICAL FEATURES OF KIDNEY TRANSPLANT RECIPIENTS WITH CYTOMEGALOVIRUS INFECTION MANIFESTATION IN THE EARLY POSTOPERATIVE PERIOD

    Directory of Open Access Journals (Sweden)

    L. V. Limareva

    2013-01-01

    Full Text Available Aim. To optimize the management of postoperative renal allograft recipients through the introduction of methods for predicting risk of manifestation of cytomegalovirus infection on the basis of a comprehensive assessment of the clinical and immunological status. Materials and methods. We retrospectively analyzed the medical records of 303 patients with end-stage renal disease, among them – were the recipients of renal allograft – 136, among whom 29 within 2 months after the operation had clinical signs of CMV infection. Assessable "CMV syndrome", laboratory evidence of CMV infection, the incidence of antigens (genes of HLA A, B and DRB *1, calculated goodness of fit χ2 and relative risk RR, changes MCP-1 in urine. Results. In renal allograft recipients with clinical and laboratory evidence of CMV infection in the early postoperative period, significantly more (χ2 > 3,8 met antigen B35. A positive association with CMV infection was detected also for DRB1 * 08, B21, B22, B41, A24 (9, B51 (5, DRB1*14 and DRB1*15. Protective effects possessed antigens / alleles of genes A26 (10, B14, B38 (16 B61 (40 and DRB1*16. MCP-1 levels in this group of recipients were raised to 2174,7 ± 296,3 pg/ml with a strong negative correlation with the levels of urea and creatinine in serum (r = 0,9, p < 0.001. Conclusion. Immunological markers of risk manifestation of CMV infection in recipients of kidneys in the early postoperative period are: the carriage of В35 и В55,56(22, В49(21, В41, DRB1*08 и DRB1*15, an increase of levels of MCP-1 in urine without increasing the levels of urea and creatinine in the serum. 

  14. Long-term results of oral valganciclovir for treatment of anterior segment inflammation secondary to cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Wong VW

    2012-04-01

    Full Text Available Victoria WY Wong, Carmen KM Chan, Dexter YL Leung, Timothy YY LaiDepartment of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, Hong Kong, People's Republic of ChinaBackground: The purpose of this study was to assess the efficacy of oral valganciclovir in the treatment of anterior segment inflammation caused by cytomegalovirus (CMV infection.Methods: Consecutive patients with anterior segment inflammation due to CMV causing anterior uveitis or corneal endotheliitis treated with oral valganciclovir were reviewed. Diagnosis of CMV infection was confirmed by polymerase chain reaction of the aqueous aspirate prior to commencement of oral valganciclovir. All patients were treated with an oral loading dose of 900 mg valganciclovir twice daily for at least 2 weeks, followed by an additional 450 mg valganciclovir twice-daily maintenance therapy. Changes in visual acuity, intraocular pressure (IOP, use of antiglaucomatous eye drops, and recurrence were analyzed.Results: Thirteen eyes of 11 patients were followed for a mean of 17.2 months. Two patients had bilateral corneal endotheliitis. All eyes had absence of anterior segment inflammation within 3 weeks after treatment. Following treatment, the mean logMAR visual acuity improved significantly from 0.58 at baseline to 0.37 at the last follow-up (P = 0.048. The mean IOP and number of antiglaucomatous eye drops also decreased significantly (P = 0.021 and P = 0.004, respectively. Five (38.5% eyes had recurrence of anterior uveitis after valganciclovir was stopped and required retreatment with oral valganciclovir.Conclusion: Oral valganciclovir appeared to be effective in controlling CMV anterior uveitis, resulting in visual improvement and IOP reduction following control of inflammation. However, despite the initial clinical response in all cases, recurrence after cessation of oral valganciclovir could occur.Keywords: cytomegalovirus infection, inflammation

  15. Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Xia, E-mail: zhai_xia_cool@126.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Qin, Ying, E-mail: qinyinggaofeng@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Chen, Yang, E-mail: cy_hmu@126.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Lin, Lexun, E-mail: linlexun@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wang, Tianying, E-mail: wangty0929@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhong, Xiaoyan, E-mail: littlerock712@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wu, Xiaoyu, E-mail: xiaoyu_wu2006@163.com [Department of Cardiology, The First Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Chen, Sijia, E-mail: chensj0802@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Li, Jing, E-mail: jing070822@163.com [Center of Electron Microscopy, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wang, Yan, E-mail: wangyan@hrbmu.edu.cn [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Fengmin, E-mail: fengminzhang@ems.hrbmu.edu.cn [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhao, Wenran, E-mail: zhaowenran2002@aliyun.com [Department of Cell Biology, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); and others

    2016-12-10

    Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection. - Highlights: • CVB3 replication induced autophagosome assembly in primary cardiac fibroblasts. • Both IL-6 and TNF-α in cardiac fibroblasts infected by CVB3 were increased. • IL-6 and TNF-α were reduced in cardiac fibroblasts when autophagy was inhibited. • Autophagosome assembly in cardiac fibroblasts of CVB-infected mice was increased.

  16. Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo

    International Nuclear Information System (INIS)

    Zhai, Xia; Qin, Ying; Chen, Yang; Lin, Lexun; Wang, Tianying; Zhong, Xiaoyan; Wu, Xiaoyu; Chen, Sijia; Li, Jing; Wang, Yan; Zhang, Fengmin; Zhao, Wenran

    2016-01-01

    Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection. - Highlights: • CVB3 replication induced autophagosome assembly in primary cardiac fibroblasts. • Both IL-6 and TNF-α in cardiac fibroblasts infected by CVB3 were increased. • IL-6 and TNF-α were reduced in cardiac fibroblasts when autophagy was inhibited. • Autophagosome assembly in cardiac fibroblasts of CVB-infected mice was increased.

  17. Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress

    International Nuclear Information System (INIS)

    Faria, Gisele; Cardoso, Cristina R.B.; Larson, Roy E.; Silva, Joao S.; Rossi, Marcos A.

    2009-01-01

    Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker of activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug

  18. Ergosterol peroxide from Cordyceps cicadae ameliorates TGF-β1-induced activation of kidney fibroblasts.

    Science.gov (United States)

    Zhu, Rong; Zheng, Rong; Deng, Yueyi; Chen, Yiping; Zhang, Shuwei

    2014-02-15

    Chronic kidney disease is a growing public health problem with an urgent need for new pharmacological agents. Ergosterol peroxide (EP) is the major sterol produced by Cordyceps cicadae Shing (C. cicadae), a widely used traditional Chinese medicine. C. cicadae has been used to treat many kinds of diseases and has a potential benefit on renoprotection. This study aimed to investigate the anti-fibrotic effects of EP as well as the underlying mechanisms. A normal rat kidney fibroblast cell line (NRK-49F) was stimulated to undergo fibroblast activation by transforming growth factor-β1 (TGF-β1) and EP treatment was applied to explore its potential anti-fibrotic effects. Cell proliferation was investigated using MTT analysis. Fibrosis-associated protein expression was analyzed using immunohistochemistry and/or Western blotting. EP treatment attenuated TGF-β1-induced renal fibroblast proliferation, expression of cytoskeleton protein and CTGF, as well as ECM production. Additionally, EP blocked TGF-β1-stimulated phosphorylation of ERK1/2, p38 and JNK pathway. Moreover, the TGF-β1-induced expression of fibronectin was attenuated by either inhibition of MAPKs or by EP treatment. In conclusion, our findings demonstrate that EP is able to suppress TGF-β1-induced fibroblasts activation in NRK-49F. This new information provides a line of theoretical evidence supporting the use of C. cicadae in the intervention of kidney disease and suggests that EP has the potential to be developed as a therapeutic agent to prevent renal fibrosis. Copyright © 2013 Elsevier GmbH. All rights reserved.

  19. Neuropathogenesis of Congenital Cytomegalovirus Infection: Disease Mechanisms and Prospects for Intervention

    OpenAIRE

    Cheeran, Maxim C.-J.; Lokensgard, James R.; Schleiss, Mark R.

    2009-01-01

    Congenital cytomegalovirus (CMV) infection is the leading infectious cause of mental retardation and hearing loss in the developed world. In recent years, there has been an improved understanding of the epidemiology, pathogenesis, and long-term disabilities associated with CMV infection. In this review, current concepts regarding the pathogenesis of neurological injury caused by CMV infections acquired by the developing fetus are summarized. The pathogenesis of CMV-induced disabilities is con...

  20. Transient Antiphospholipid Syndrome Associated with Primary Cytomegalovirus Infection: A Case Report and Literature Review

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Nakayama

    2014-01-01

    Full Text Available Viral infection is known to induce transient autoimmunity in humans. Acute cytomegalovirus (CMV infection is implicated in occasional thrombosis formation. We here, for the first time, report a 19-year-old female who had an acute CMV infection, leading to a deep venous thrombosis and a pulmonary embolism along with transient appearance of lupus anticoagulant. The pathological role of antiphospholipid antibodies in CMV-mediated thrombosis is discussed.

  1. FEL induced molecular operation on cultured fibroblast and cholesterol ester

    International Nuclear Information System (INIS)

    Awazu, Kunio; Ogino, Seiji; Nishimura, Eiichi; Tomimasu, Takio; Yasumoto, Masato.

    1997-01-01

    Free Electron Lasers can be used to molecular operation such as the delivery of a number of molecules into cells or the separation of cholesterol ester. First, cultured NIH3T3 cells are exposed to high-intensity short pulse Free Electron Laser (FEL). The FEL is tuned to an absorption maximum wavelength, 6.1 μm, which was measured by microscopic FTIR. A fluorescence dye in the cell suspension is more absorbed into the cell with the FEL exposure due to the FEL-induced mechanical stress to the cell membrane. A quantitative fluorescence microscopy is used to determine the efficiency of delivery. Second, as a compound in a lipid cell, cholesterol ester was exposed to 5.75 μm FEL. FTIR measurement was done to evaluate the modification of the cholesterol ester. The result showed that the fluorescence intensity of sample cells were higher than that of control cells, and there was significant difference between the control and the sample group. Blebbing and the colony formation of the cells were observed for cells with mechanical stress. As for the cholesterol ester, it can be modified by the FEL irradiation. These results showed that FEL can be used as a molecular operational tool by photo-chemical and photo-mechanical interaction. (author)

  2. Portulaca oleracea extracts protect human keratinocytes and fibroblasts from UV-induced apoptosis.

    Science.gov (United States)

    Lee, Suyeon; Kim, Ki Ho; Park, Changhoon; Lee, Jong-Suk; Kim, Young Heui

    2014-10-01

    Portulaca oleracea extracts, known as Ma Chi Hyun in the traditional Korean medicine, show a variety of biomedical efficacies including those in anti-inflammation and anti-allergy. In this study, we investigate the protective activity of the P. oleracea extracts against UVB-induced damage in human epithelial keratinocytes and fibroblasts by several apoptosis-related tests. The results suggest that P. oleracea extracts have protective effects from UVB-induced apoptosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

    Science.gov (United States)

    Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

    2007-05-01

    Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

  4. Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

    International Nuclear Information System (INIS)

    Hoever, Gerold; Vogel, Jens-Uwe; Lukashenko, Polina; Hofmann, Wolf-Karsten; Komor, Martina; Doerr, Hans Wilhelm; Cinatl, Jindrich

    2005-01-01

    In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies

  5. Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

    Directory of Open Access Journals (Sweden)

    Yinghua Song

    2013-10-01

    Full Text Available OBJECTIVE: To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice. METHODS: Skin fibroblasts that were isolated from three systemic scleroderma (SSc patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM. Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I procollagen (COL1A1, alpha 1 (III procollagen (COL3A1, matrix metalloproteinase (MMP-1 and tissue inhibitor of matrix metalloproteinase (TIMP-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA. Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR. RESULTS: Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3. CONCLUSION: Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a

  6. Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

    NARCIS (Netherlands)

    Scharstuhl, A.; Mutsaers, H.A.M.; Pennings, S.W.C.; Szarek, W.A.; Russel, F.G.M.; Wagener, F.A.D.T.G.

    2009-01-01

    Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and

  7. Specific and Non-Specific Factors of Humoral Immunity as Markers for Pregnancy Loss in Women with Cytomegalovirus Infection

    Directory of Open Access Journals (Sweden)

    Irina A. Andrievskaya

    2015-12-01

    Full Text Available The aim of this study was to estimate the changes in humoral immunity and their association with complications of pregnancy (spontaneous abortions, threatened miscarriage, premature birth depending on the gestational age and recurrence of cytomegalovirus infection (CMVI. A direct relationship between the frequency of detection of an anti-CMV IgG antibody titer of 1:1600 and the prevalence of acute respiratory disease during pregnancy has been identified. We found an imbalance in the production of the non-specific antibodies (an increase in the blood levels of total IgM and a decrease in IgA and IgG levels in the subgroup of women with relapsed CMVI at 6 to 8 weeks of gestation and spontaneous abortion, as well as in the subgroup of women with relapsed CMVI at 15 to 21 weeks of gestation and the risk of the late miscarriage, compared to those with relapsed CMVI at 9 to 14 weeks and 22 to 32 weeks of gestation. An increase in blood levels of total IgM and IgG and a decrease in IgA level was identified in the subgroup of women with relapsed CMVI at 9 to14 weeks of gestation and a threatened abortion, as well as in the subgroup of women with relapsed CMVI at 22 to 32 weeks of gestation and preterm birth. The obtained data of the imbalance in the primary and secondary immune response in CMV- seropositive pregnant women during relapsed CMVI indicate disturbances in the systemic and local intercellular interactions of immunocompetent cells, which lead to an imbalance in the production of antibodies involved in the elimination of viral agents and to the development of a systemic inflammatory response that complicates the course of pregnancy. CMVI relapse at 7 to 8 weeks of gestation is associated with reproductive losses; a risk for threatened miscarriage, threatened premature labor, and retrochorial hematoma increases significantly with CMVI relapse in the more remote gestational age.

  8. Is cytomegalovirus infection related to inflammatory bowel disease, especially steroid-resistant inflammatory bowel disease? A meta-analysis

    Directory of Open Access Journals (Sweden)

    Lv Y

    2017-12-01

    Full Text Available Ya-li Lv, Fei-fei Han, Yang-jie Jia, Zi-rui Wan, Li-li Gong, He Liu, Li-hong Liu Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China Background: Human cytomegalovirus (HCMV infection has been associated with inflammatory bowel disease (IBD. Numerous studies have been conducted to analyze the association between HCMV infection and risk of IBD and steroid-resistant IBD, but no clear consensus had been reached. Objectives: The aim of this study was to confirm this relationship precisely by doing a systematic review and meta-analysis. Study design: We identified relevant studies through a search of PubMed and Embase. Studies were eligible for inclusion if they 1 evaluated the association between HCMV infection and IBD disease; 2 evaluated the association between HCMV infection and steroid-resistant IBD disease; 3 were case–control studies or nested case–control studies; 4 provided the numbers (or percentage of positivity for HCMV infection in cases and controls, respectively. Data were extracted and analyzed independently by two investigators. Results and conclusion: A total of 18 studies including 1,168 patients and 951 health groups was identified, and HCMV infection was distinctly confirmed as a risk factor for the occurrence and development of IBD. When involving 17 studies including 1,306 IBD patients, a total of 52.9% of patients in the cytomegalovirus (CMV-positive groups were observed to have steroid resistance, compared with 30.2% of patients in the CMV-negative groups. There was a significant difference in the risk of steroid resistance between people exposed to HCMV infection and those not exposed HCMV infection in IBD patients. This meta-analysis suggested that HCMV infection is associated with an increased risk for IBD and steroid-resistant IBD. Keywords: cytomegalovirus, infection, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, meta-analysis

  9. Melatonin suppresses acrolein-induced IL-8 production in human pulmonary fibroblasts.

    Science.gov (United States)

    Kim, Gun-Dong; Lee, Seung Eun; Kim, Tae-Ho; Jin, Young-Ho; Park, Yong Seek; Park, Cheung-Seog

    2012-04-01

    Cigarette smoke (CS) causes harmful alterations in the lungs and airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). In addition to COPD, active cigarette smoking causes other respiratory diseases and diminishes health status. Furthermore, recent studies show that, α, β-unsaturated aldehyde acrolein in CS induces the production of interleukin (IL)-8, which is known to be related to bronchitis, rhinitis, pulmonary fibrosis, and asthma. In addition, lung and pulmonary fibroblasts secrete IL-8, which has a chemotactic effect on leukocytes, and which in turn, play a critical role in lung inflammation. On the other hand, melatonin regulates circadian rhythm homeostasis in humans and has many other effects, which include antioxidant and anti-inflammatory effects, as demonstrated by the reduced expressions of iNOS, IL-1β, and IL-6 and increased glutathione (GSH) and superoxide dismutase activities. In this study, we investigated whether melatonin suppresses acrolein-induced IL-8 secretion in human pulmonary fibroblasts (HPFs). It was found that acrolein-induced IL-8 production was accompanied by increased levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK1/2) in HPFs, and that melatonin suppressed IL-8 production in HPFs. These results suggest that melatonin suppresses acrolein-induced IL-8 production via ERK1/2 and phosphatidylinositol 3-kinase (PI3K)/Akt signal inhibition in HPFs. © 2011 John Wiley & Sons A/S.

  10. Cinnamaldehyde impairs high glucose-induced hypertrophy in renal interstitial fibroblasts

    International Nuclear Information System (INIS)

    Chao, Louis Kuoping; Chang, W.-T.; Shih, Y.-W.; Huang, J.-S.

    2010-01-01

    Cinnamaldehyde is a major and a bioactive compound isolated from the leaves of Cinnamomum osmophloeum kaneh. To explore whether cinnamaldehyde was linked to altered high glucose (HG)-mediated renal tubulointerstitial fibrosis in diabetic nephropathy (DN), the molecular mechanisms of cinnamaldehyde responsible for inhibition of HG-induced hypertrophy in renal interstitial fibroblasts were examined. We found that cinnamaldehyde caused inhibition of HG-induced cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, cleaved poly(ADP-ribose) polymerase (PARP) protein expression, and mitochondrial cytochrome c release in HG or cinnamaldehyde treatments in these cells. HG-induced extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) (but not the Janus kinase 2/signal transducers and activators of transcription) activation was markedly blocked by cinnamaldehyde. The ability of cinnamaldehyde to inhibit HG-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of collagen IV, fibronectin, and α-smooth muscle actin (α-SMA). The results obtained in this study suggest that cinnamaldehyde treatment of renal interstitial fibroblasts that have been stimulated by HG reduces their ability to proliferate and hypertrophy through mechanisms that may be dependent on inactivation of the ERK/JNK/p38 MAPK pathway.

  11. Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

    Science.gov (United States)

    Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

    2015-08-15

    COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Inhibition of hydrogen peroxide induced injuring on human skin fibroblast by Ulva prolifera polysaccharide.

    Science.gov (United States)

    Cai, Chuner; Guo, Ziye; Yang, Yayun; Geng, Zhonglei; Tang, Langlang; Zhao, Minglin; Qiu, Yuyan; Chen, Yifan; He, Peimin

    2016-10-01

    Ulva prolifera can protect human skin fibroblast from being injured by hydrogen peroxide. This work studied the composition of Ulva prolifera polysaccharide and identified its physicochemical properties. The results showed that the cell proliferation of 0.5mg/mL crude polysaccharide was 154.4% of that in negative control group. Moreover, ROS detection indices, including DCFH-DA, GSH-PX, MDA and CAT, indicated that crude polysaccharide could improve cellular ability to scavenge free radical and decrease the injury on human skin fibroblast by hydrogen peroxide. In purified polysaccharide, the activity of fraction P1-1 was the highest, with 174.6% of that in negative control group. The average molecular weight of P1-1 was 137kD with 18.0% of sulfate content. This work showed the inhibition of hydrogen peroxide induced injuries on human skin fibroblast by Ulva prolifera polysaccharide, which may further evaluate the application of U. prolifera on cosmetics. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Fibroblast and T cells conditioned media induce maturation dendritic cell and promote T helper immune response

    Directory of Open Access Journals (Sweden)

    Masoumeh Asadi

    2012-06-01

    Full Text Available Dendritic cells (DCs induce pathogen-specific T cell responses. We comprehensively studied the effects of addition of maturation stimulus, fibroblasts (fibroblast conditioned medium, PHA activated T cells (T cell conditioned medium, and mixture of fibroblast & PHA activated T cells (FCM-TCCM conditioned media on maturation of DCs. Monocytes were cultured with GM-CSF and IL-4 for five days. Maturation factors included MCM and TNF-α as control group. FCM and TCCM, or FCM-TCCM supernatant were considered as the treatment group. Tumor antigens were added at day five. Matured DCs were harvested at day seven. Phenotypic and functional analyses were carried out using anti (CD14, CD80, CD86, CD83 and HLA-DR monoclonal antibodies. Phagocytic activity, mixed lymphocyte reaction (MLR and cytokine production were also evaluated. At the end of culturing period, significantly fully matured DCs with large amount cytoplasm and copious dendritic projections were found in the presence of MCM, TNF-α with or without FCM, TCCM, FCM as well as TCCM. Flow cytometric analysis revealed that expression of CD14 decreased in particular in treated DCs, at the 5th day and expression of CD80, CD86 and HLA-DR was higher when FCM, TCCM, FCM plus TCCM were added to maturation factor. This study demonstrated that DCs matured with these methods had optimum function in comparison with either factor alone.

  14. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  15. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    International Nuclear Information System (INIS)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-01-01

    Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  16. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    Science.gov (United States)

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    International Nuclear Information System (INIS)

    Jiang, Shao-Yun; Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan; Deng, Jia-Yin

    2012-01-01

    Highlights: ► High glucose significantly induced TLR2 expression in gingival fibroblasts. ► High glucose increased NF-κB p65 nuclear activity, IL-1β and TNF-α levels. ► PKC-α/δ-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-κB) p65 nuclear activity, tumor necrosis factor-α (TNF-α) and interleukin-lβ (IL-1β) levels. Protein kinase C (PKC)-α and δ knockdown with siRNA significantly decreased TLR2 and NF-κB p65 expression (p < 0.05), whereas inhibition of PKC-β had no effect on TLR2 and NF-κB p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-κB expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-α and IL-1β secretion via inducing TLR2 through PKC-α and PKC-δ in human gingival fibroblasts.

  18. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shao-Yun, E-mail: jiangshaoyun@yahoo.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Deng, Jia-Yin, E-mail: yazhou2991@126.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer High glucose significantly induced TLR2 expression in gingival fibroblasts. Black-Right-Pointing-Pointer High glucose increased NF-{kappa}B p65 nuclear activity, IL-1{beta} and TNF-{alpha} levels. Black-Right-Pointing-Pointer PKC-{alpha}/{delta}-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-{kappa}B) p65 nuclear activity, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-l{beta} (IL-1{beta}) levels. Protein kinase C (PKC)-{alpha} and {delta} knockdown with siRNA significantly decreased TLR2 and NF-{kappa}B p65 expression (p < 0.05), whereas inhibition of PKC-{beta} had no effect on TLR2 and NF-{kappa}B p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-{kappa}B expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-{alpha} and IL-1{beta} secretion via inducing TLR2 through PKC-{alpha} and PKC-{delta} in human gingival fibroblasts.

  19. Congenital cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Katarina Rednak-Paradiž

    2006-11-01

    Full Text Available Background: CMV is the most common agent that causes congenital virus infection. Only 10 % of infected children have symptomatic infection immediately after birth. Signs of central nervous system damage, neurosensory deafness and delayed psychomotor development may manifest as a result of asimptomatic congenital infection later in childhood. In the article we present basic properties of CMV; we describe clinical picture of the congenital infection and possibilities of diagnose and its treatment. We present five children with symptomatic congenital CMV infection that were hospitalized for the period 1992–2002 at the Neonatal department in the University Children’s Hospital in Ljubljana.Conclusions: Identification of infected neonates, especially those with asimptomatic congenital CMV infection, is difficult. Latest incidence of infection in Slovenia is unknown. With new investigations the efficiency of antiviral therapy was discovered but exact indications for therapy are not yet known. CMV vaccine, once available, may ultimately be the best control strategy for this important public health problem. Proper educating women in childbearing age about the risks of CMV and how to avoid disease transmission during pregnancy (hand washing, avoiding mouth-to-mouth contact with preschool children, usage of gloves especially when handling dipers or respiratory secretions are the only control strategies available.

  20. Cytomegalovirus infection with lissencephaly

    Directory of Open Access Journals (Sweden)

    Joseph Leena

    2008-07-01

    Full Text Available Lissencephaly is a malformation of the brain in which the brain surface is smooth, rather than convoluted. Among the various causes of lissencephaly, infection by a virus during pregnancy plays an important role. Cytomegalovirus (CMV is an important pathogen causing this anomaly. We present this case of a young female with 24-week-gestation diagnosed on ultrasound as carrying an anomalous fetus with lissencephalic features. At autopsy, there were multiple intra-nuclear CMV inclusions in the brain and the kidneys. This case is presented for its rarity and for the documentation of the tissue localization of CMV inclusions at autopsy.

  1. Butyrate down regulates BCL-XL and sensitizes human fibroblasts to radiation and chemotherapy induced apoptosis

    International Nuclear Information System (INIS)

    Chung, Diana H.; Ljungman, Mats; Zhang Fenfen; Chen Feng; McLaughlin, William P.

    1997-01-01

    Purpose/Objective: Butyrate is a short chain fatty acid that has been implicated in the induction of cell cycle arrest, cell differentiation and apoptosis. The purpose of this study was to determine if butyrate treatment sensitizes cells to radiation or chemotherapy induced apoptosis. Materials and Methods: Normal neonatal human diploid fibroblasts were used throughout this study. Apoptosis was scored and quantified using three different methods. First, cell morphology using propidium iodide and fluorescence microscopy was used to qualitatively determine apoptosis and to quantify the percentage of cells undergoing apoptosis. Second, apoptosis induced DNA degradation was scored by quantifying the amount of cells appearing in a sub-G1 peak using fixed and PI-stained cells and flow cytometry. Third, apoptosis-induced DNA degradation was examined by using an assay involving direct lysis of cells in the wells of agarose gels followed by conventional gel electrophoresis. Western blotting was used to quantify the cellular levels of the apoptosis regulators, Bcl-2, Bcl-XL and Bax. Results: Human diploid fibroblasts, which were resistant to radiation induced apoptosis, were found to undergo massive apoptosis when radiation was combined with butyrate treatment. Sensitization was obtained when butyrate was added before or after radiation although the combination of both pre and post-treatment was the most effective. Butyrate was also found to enhance UV light and cisplatin-induced apoptosis. These findings correlated with a reduction of the apoptosis antagonist Bcl-XL. Bcl-XL levels significantly dropped in a time and dose dependent manner. In addition, butyrate effectively blocked UV-induced accumulation of p53. Conclusion: Our results suggest that butyrate may be an attractive agent to use in combination with radiation or chemotherapy to lower the apoptotic threshold of tumor cells, regardless of the p53 status of the tumor cells

  2. Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

    Science.gov (United States)

    Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

    2013-01-01

    Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID

  3. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yulianti

    2015-08-01

    Full Text Available Asiatiocoside, a saponin component isolated from Centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (HDF and synthesis of collagen. The skin-renewing cells and type I and III collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. Usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. The aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type I and III collagen synthesis in human dermal fibroblast. Methods Laboratory experiments were conducted using human dermal fibroblasts (HDF isolated from human foreskin explants. Seven passages of HDF were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. Cell viability in all groups was tested with the MTT assay to assess HDF proliferation. Type I and III collagen synthesis was examined using the respective ELISA kits. Analysis of variance was performed to compare the treatment groups. Results Asiaticoside had significantly stronger effects on HDF proliferation than retinoic acid (p<0.05. The type III collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05. Conclusion Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  4. Heat Shock Protein 90 Inhibitor (17-AAG) Induces Apoptosis and Decreases Cell Migration/Motility of Keloid Fibroblasts.

    Science.gov (United States)

    Yun, In Sik; Lee, Mi Hee; Rah, Dong Kyun; Lew, Dae Hyun; Park, Jong-Chul; Lee, Won Jai

    2015-07-01

    The regulation of apoptosis, proliferation, and migration of fibroblasts is altered in keloids. The 90-kDa heat shock protein (heat shock protein 90) is known to play a key role in such regulation. Therefore, the authors investigated whether the inhibition of heat shock protein 90 in keloid fibroblasts could induce apoptosis and attenuate keloid fibroblast proliferation and migration. The authors evaluated heat shock protein 90 expression in keloid tissues with immunohistochemistry. The authors used cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and annexin V/propidium iodide staining for apoptosis, a wound healing model and cell tracking system to assess cell migration, and Akt Western blotting analysis in keloid fibroblasts after inhibition of heat shock protein 90 with 17-allylaminodemethoxygeldanamycin (17-AAG). The expression of heat shock protein 90 in keloid tissues was significantly increased compared with normal tissues. The 17-AAG-treated keloid fibroblasts showed significantly decreased proliferation, promotion of apoptosis, and decreased expression of Akt. Furthermore, a dose-dependent decrease in cell migration was noted after 17-AAG treatment of keloid fibroblasts. The 17-AAG-treated keloid fibroblasts had less directionality to the wound center and migrated a shorter distance. The authors confirmed that the inhibition of heat shock protein 90 in keloid fibroblasts could promote apoptosis and attenuate proliferation and migration of keloid fibroblasts. Therefore, the authors think that the inhibition of heat shock protein 90 is a key factor in the regulation of biological processes in keloids. With further preclinical study, the authors will be able to apply these results clinically for keloid treatment.

  5. The role of γ-ray-induced fibroblast apoptosis in inhibiting biliary duct hypertrophic scar formation in dogs

    International Nuclear Information System (INIS)

    He Guijin; Zhang Hong; Gao Xinyi; Xu Shuhe; Gao Hong; Jiang Weiguo; Jiangtao; Dai Xianwei; Ma Kai

    2005-01-01

    Objective: To investigate the role of γ-ray-induced fibroblast apoptosis in the inhibition of biliary duct hypertrophic scar formation in dogs. Methods: γ-radiation-induced apoptotic fibroblast cells were analysed by using transmission electron microscopy and DNA from frozen biliary duct tissue was extracted with phenol chloroform. DNA ladder profile after extraction of RNA was observed, and apoptosis cells in paraffinem-bedded biliary duct tissue sections were examined used immuno-histochemical method. Dog biliary duct cross-sections were stained with hematoxylin-erosin, Masson's trichrome, and Verhoeff-van Giesen stains. Muscle formation area, lumen circumference, and stenosis degree were determined by a computer-assisted image analysis system. Results: 103 Pd radioactive stent significantly inhibited fibroblast proliferation. The features of fibroblast apoptosis (e.g, apoptic bodies, DNA ladder band) could be seen in the 103 Pd radioactive stent group. The fibroblast apoptotic rate was significantly increased in the 103 Pd radioactive stent group than in the control group (P 103 Pd radioactive stent significantly reduced biliary muscular formation. Conclusion: 103 Pd radioactive stent could have the effect of inhibiting the proliferation of scar-forming fibroblast, and thus could be used for treatment and (or) prevention of hypertrophic scar formation in biliary duct. (authors)

  6. Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

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    Karin Zins

    2014-01-01

    Full Text Available The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

  7. Small molecular antioxidants effectively protect from PUVA-induced oxidative stress responses underlying fibroblast senescence and photoaging.

    Science.gov (United States)

    Briganti, Stefania; Wlaschek, Meinhard; Hinrichs, Christina; Bellei, Barbara; Flori, Enrica; Treiber, Nicolai; Iben, Sebastian; Picardo, Mauro; Scharffetter-Kochanek, Karin

    2008-09-01

    Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.

  8. Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Ritter, M.A.; Williams, J.R.

    1981-01-01

    Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent lethality. (Auth.)

  9. The molecular mechanism of leptin secretion and expression induced by aristolochic acid in kidney fibroblast.

    Directory of Open Access Journals (Sweden)

    Tsung-Chieh Lin

    Full Text Available BACKGROUND: Leptin is a peptide hormone playing pivotal role in regulating food intake and energy expenditure. Growing evidence has suggested the pro-inflammatory and fibrogenic properties of leptin. In addition, patients with renal fibrosis have higher level of plasma leptin, which was due to the increased leptin production. Aristolochic acid (AA is a botanical toxin characterized to associate with the development of renal fibrosis including tubulointerstitial fibrosis. However, whether leptin is upregulated to participate in AA-induced kidney fibrosis remain completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, leptin expression was increased by sublethal dose of AA in kidney fibroblast NRK49f determined by enzyme-linked immunosorbent assay and Western blot. Data from real-time reverse transcriptase-polymerase chain reaction revealed that leptin was upregulated by AA at transcriptional level. DNA binding activity of CCAAT enhancer binding protein α (C/EBP α, one of the transcription factors for leptin gene, was enhanced in DNA affinity precipitation assay and chromatin immunoprecipitation experiments. Knockdown of C/EBP α expression by small interfering RNA markedly reduced AA-induced leptin expression. Moreover, AA promoted Akt interaction with p-PDK1, and increased phosphorylated activation of Akt. Akt knockdown, and inhibition of Akt signaling by LY294002 and mTOR inhibitor rapamycin reduced leptin expression. Furthermore, treatment of LY294002 or rapamycin significantly suppressed AA-induced C/EBP α DNA-binding activity. These results suggest that Akt and C/EBP α activation were involved in AA-regulated leptin expression. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the first that AA could induce secretion and expression of fibrogenic leptin in kidney fibroblasts, which reveal potential involvement of leptin in the progression of kidney fibrosis in aristolochic acid nephropathy.

  10. Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

    Science.gov (United States)

    Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2012-04-10

    Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

  11. Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

    Science.gov (United States)

    Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2011-06-01

    Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

  12. Acetylsalicylic acid inhibits IL-18-induced cardiac fibroblast migration through the induction of RECK.

    Science.gov (United States)

    Siddesha, Jalahalli M; Valente, Anthony J; Sakamuri, Siva S V P; Gardner, Jason D; Delafontaine, Patrice; Noda, Makoto; Chandrasekar, Bysani

    2014-07-01

    The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS-dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a unique membrane-anchored MMP regulator, on IL-18-induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL-18-induced migration of primary mouse CF was dependent on both IKK/NF-κB- and JNK/AP-1-mediated MMP9 induction and Sp1-mediated RECK suppression, mechanisms that required Nox4-dependent H(2)O(2) generation. Notably, forced expression of RECK attenuated IL-18-induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL-18-induced H(2)O(2) generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL-18-induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms. © 2013 Wiley Periodicals, Inc.

  13. Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

    Science.gov (United States)

    Li, Xiang; Liu, Defang; Ma, Yantao; Du, Xiaomin; Jing, Junzhan; Wang, Lipeng; Xie, Bingqing; Sun, Da; Sun, Shaoqiang; Jin, Xueqin; Zhang, Xu; Zhao, Ting; Guan, Jingyang; Yi, Zexuan; Lai, Weifeng; Zheng, Ping; Huang, Zhuo; Chang, Yanzhong; Chai, Zhen; Xu, Jun; Deng, Hongkui

    2017-08-03

    Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    Directory of Open Access Journals (Sweden)

    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  15. Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

    Directory of Open Access Journals (Sweden)

    Min Ji Choi

    2015-06-01

    Full Text Available Transforming growth factor-β1 (TGF-β1 has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD. The mothers against decapentaplegic homolog 7 (SMAD7 is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator and induced the expression of poly (ADP-ribose polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

  16. The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

    Science.gov (United States)

    Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

    2014-01-01

    Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

  17. Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts

    DEFF Research Database (Denmark)

    Dunlevy, J R; Couchman, J R

    1995-01-01

    Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8...

  18. Photodynamic therapy induces antifibrotic alterations in primary human vocal fold fibroblasts.

    Science.gov (United States)

    Zhang, Chi; Wang, Jiajia; Chou, Adriana; Gong, Ting; Devine, Erin E; Jiang, Jack J

    2018-04-18

    Photodynamic therapy (PDT) is a promising treatment modality for laryngeal dysplasia, early-stage carcinoma, and papilloma, and was reported to have the ability to preserve laryngeal function and voice quality without clinical fibrotic response. We aimed to investigate the mechanism behind the antifibrotic effects of PDT on primary human vocal fold fibroblasts (VFFs) in vitro. In vitro analysis from one human donor. Cell viability of VFFs in response to varying doses of PDT was investigated by the Cell Counting Kit-8 method. Sublethal-dose PDT (SL-PDT) was used for the following experiments. Expression of genes related to vocal fold extracellular matrix formation was analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Effects of PDT on cell migration, collagen contraction, and transforming growth factor β-1 (TGF-β1)-induced myofibroblast differentiation were also analyzed. PDT affects the viability of VFFs in a dose-dependent manner. SL-PDT significantly changed the expression profile of VFFs with antifibrotic effects. It also inhibited cell migration, reduced collagen contraction, and reversed the fibroblast-myofibroblast differentiation induced by TGF-β1. SL-PDT induces antifibrotic alterations in VFFs. This could explain the low incidence of vocal fold scar associated with PDT. Moreover, PDT may be useful in treating existing vocal fold scars. Further studies should focus on the in vivo effect of PDT on vocal fold wound healing and scar remodeling. NA Laryngoscope, 2018. © 2018 The American Laryngological, Rhinological and Otological Society, Inc.

  19. Clarithromycin attenuates IL-13–induced periostin production in human lung fibroblasts

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    Kosaku Komiya

    2017-02-01

    Full Text Available Abstract Background Periostin is a biomarker indicating the presence of type 2 inflammation and submucosal fibrosis; serum periostin levels have been associated with asthma severity. Macrolides have immunomodulatory effects and are considered a potential therapy for patients with severe asthma. Therefore, we investigated whether macrolides can also modulate pulmonary periostin production. Methods Using quantitative PCR and ELISA, we measured periostin production in human lung fibroblasts stimulated by interleukin-13 (IL-13 in the presence of two 14-member–ring macrolides—clarithromycin or erythromycin—or a 16-member–ring macrolide, josamycin. Phosphorylation of signal transducers and activators of transcription 6 (STAT6, downstream of IL-13 signaling, was evaluated by Western blotting. Changes in global gene expression profile induced by IL-13 and/or clarithromycin were assessed by DNA microarray analysis. Results Clarithromycin and erythromycin, but not josamycin, inhibited IL-13–stimulated periostin production. The inhibitory effects of clarithromycin were stronger than those of erythromycin. Clarithromycin significantly attenuated STAT6 phosphorylation induced by IL-13. Global gene expression analyses demonstrated that IL-13 increased mRNA expression of 454 genes more than 4-fold, while decreasing its expression in 390 of these genes (85.9%, mainly “extracellular,” “plasma membrane,” or “defense response” genes. On the other hand, clarithromycin suppressed 9.8% of the genes in the absence of IL-13. Clarithromycin primarily attenuated the gene expression of extracellular matrix protein, including periostin, especially after IL-13. Conclusions Clarithromycin suppressed IL-13–induced periostin production in human lung fibroblasts, in part by inhibiting STAT6 phosphorylation. This suggests a novel mechanism of the immunomodulatory effect of clarithromycin in asthmatic airway inflammation and fibrosis.

  20. Upregulation of NOXA by 10-Hydroxycamptothecin plays a key role in inducing fibroblasts apoptosis and reducing epidural fibrosis

    Directory of Open Access Journals (Sweden)

    Jihang Dai

    2017-01-01

    Full Text Available The fibrosis that develops following laminectomy or discectomy often causes serious complications, and the proliferation of fibroblasts is thought to be the major cause of epidural fibrosis. 10-Hydroxycamptothecin (HCPT has been proven to be efficient in preventing epidural fibrosis, but the exact mechanism is still unclear. NOXA is a significant regulator of cell apoptosis, which has been reported to be beneficial in the treatment of fibrosis. We performed a series of experiments, both in vitro and in vivo, to explore the intrinsic mechanism of HCPT that underlies the induction of apoptosis in fibroblasts, and also to investigate whether HCPT has positive effects on epidural fibrosis following laminectomy in rats. Fibroblasts were cultured in vitro and stimulated by varying concentrations of HCPT (0, 1, 2, 4 µg/ml for various durations (0, 24, 48, 72 h; the effect of HCPT in inducing the apoptosis of fibroblasts was investigated via Western blots and TUNEL assay. Our results showed that HCPT could induce apoptosis in fibroblasts and up-regulate the expression of NOXA. Following the knockdown of NOXA in fibroblasts, the results of Western blot analysis showed that the level of apoptotic markers, such as cleaved-PARP and Bax, was decreased. The results from the TUNEL assay also showed a decreased rate of apoptosis in NOXA-knocked down fibroblasts. For the in vivo studies, we performed a laminectomy at the L1-L2 levels in rats and applied HCPT of different concentrations (0.2, 0.1, 0.05 mg/ml and saline locally; the macroscopic histological assessment, hydroxyproline content analysis and histological staining were performed to evaluate the effect of HCPT on reducing epidural fibrosis. The TUNEL assay in epidural tissues showed that HCPT could obviously induce apoptosis in fibroblasts in a dose-dependent manner. Also, immunohistochemical staining showed that the expression of NOXA increased as the concentrations of HCPT increased. Our findings are

  1. Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage

    Directory of Open Access Journals (Sweden)

    Claudia von Montfort

    2015-04-01

    Full Text Available Recently, it has been published that cerium (Ce oxide nanoparticles (CNP; nanoceria are able to downregulate tumor invasion in cancer cell lines. Redox-active CNP exhibit both selective pro-oxidative and antioxidative properties, the first being responsible for impairment of tumor growth and invasion. A non-toxic and even protective effect of CNP in human dermal fibroblasts (HDF has already been observed. However, the effect on important parameters such as cell death, proliferation and redox state of the cells needs further clarification. Here, we present that nanoceria prevent HDF from reactive oxygen species (ROS-induced cell death and stimulate proliferation due to the antioxidative property of these particles.

  2. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

    2007-01-01

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

  3. Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

    International Nuclear Information System (INIS)

    Ma, Jane; Bishoff, Bridget; Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane; Castranova, Vincent

    2017-01-01

    The emission of cerium oxide nanoparticles (CeO 2 ) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO 2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO 2 -induced fibrosis. Male Sprague-Dawley rats were exposed to CeO 2 (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO 2 (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO 2 -exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO 2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO 2 -exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO 2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO 2 nanoparticle exposure. - Highlights: • CeO 2 exposure induced lung fibrosis. • CeO 2 were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO 2 caused ATII cell hypertrophy and hyperplasia and altered fibroblast function

  4. Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Jane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Bishoff, Bridget [Mylan Pharmaceuticals, Morganntown, WV (United States); Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Castranova, Vincent, E-mail: vcastran@hsc.wvu.edu [School of Pharmacy, West Virginia University, Morgantown, WV (United States)

    2017-05-15

    The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII

  5. SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

    International Nuclear Information System (INIS)

    Liesenfeld, Melanie; Mosig, Sandy; Funke, Harald; Jansen, Lars; Runnebaum, Ingo B; Dürst, Matthias; Backsch, Claudia

    2013-01-01

    Genetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer. Microarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction. Overall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro. The relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence

  6. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Lerner, Chad A.; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K.; Elder, Alison; Rahman, Irfan, E-mail: irfan_rahman@urmc.rochester.edu

    2016-09-02

    Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping. - Graphical abstract: Oxidants and possibly reactive properties of metal particles in E-cig aerosols impart mitochondrial oxidative stress and DNA damage. These biological effects accompany inflammatory response which may raise concern regarding long term E-cig use. Mitochondria may be particularly sensitive to reactive properties of E-cig aerosols in addition to the potential for them to induce genotoxic stress by generating increased ROS. - Highlights: • Mitochondria are sensitive to both E-cig aerosols and metal nanoparticles. • Increased mtROS by E-cig aerosol is associated with disrupted mitochondrial energy. • E-cig causes nuclear DNA fragmentation. • E-cig aerosols induce pro-inflammatory response in human fibroblasts.

  7. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts

    International Nuclear Information System (INIS)

    Lerner, Chad A.; Rutagarama, Pierrot; Ahmad, Tanveer; Sundar, Isaac K.; Elder, Alison; Rahman, Irfan

    2016-01-01

    Oxidants or nanoparticles have recently been identified as constituents of aerosols released from various styles of electronic cigarettes (E-cigs). Cells in the lung may be directly exposed to these constituents and harbor reactive properties capable of incurring acute cell injury. Our results show mitochondria are sensitive to both E-cig aerosols and aerosol containing copper nanoparticles when exposed to human lung fibroblasts (HFL-1) using an Air-Liquid Interface culture system, evident by elevated levels of mitochondrial ROS (mtROS). Increased mtROS after aerosol exposure is associated with reduced stability of OxPhos electron transport chain (ETC) complex IV subunit and nuclear DNA fragmentation. Increased levels of IL-8 and IL-6 in HFL-1 conditioned media were also observed. These findings reveal both mitochondrial, genotoxic, and inflammatory stresses are features of direct cell exposure to E-cig aerosols which are ensued by inflammatory duress, raising a concern on deleterious effect of vaping. - Graphical abstract: Oxidants and possibly reactive properties of metal particles in E-cig aerosols impart mitochondrial oxidative stress and DNA damage. These biological effects accompany inflammatory response which may raise concern regarding long term E-cig use. Mitochondria may be particularly sensitive to reactive properties of E-cig aerosols in addition to the potential for them to induce genotoxic stress by generating increased ROS. - Highlights: • Mitochondria are sensitive to both E-cig aerosols and metal nanoparticles. • Increased mtROS by E-cig aerosol is associated with disrupted mitochondrial energy. • E-cig causes nuclear DNA fragmentation. • E-cig aerosols induce pro-inflammatory response in human fibroblasts.

  8. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

    Directory of Open Access Journals (Sweden)

    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

  9. Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts

    International Nuclear Information System (INIS)

    Xinbo Cheng; Nge Cheong; Ya Wang; Iliakis, George

    1996-01-01

    Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or γ-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21 cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

  10. Fibroblast Growth Factor 21 Deficiency Attenuates Experimental Colitis-Induced Adipose Tissue Lipolysis

    Directory of Open Access Journals (Sweden)

    Liming Liu

    2017-01-01

    Full Text Available Aims. Nutrient deficiencies are common in patients with inflammatory bowel disease (IBD. Adipose tissue plays a critical role in regulating energy balance. Fibroblast growth factor 21 (FGF21 is an important endocrine metabolic regulator with emerging beneficial roles in lipid homeostasis. We investigated the impact of FGF21 in experimental colitis-induced epididymal white adipose tissue (eWAT lipolysis. Methods. Mice were given 2.5% dextran sulfate sodium (DSS ad libitum for 7 days to induce colitis. The role of FGF21 was investigated using antibody neutralization or knockout (KO mice. Lipolysis index and adipose lipolytic enzymes were determined. In addition, 3T3-L1 cells were pretreated with IL-6, followed by recombinant human FGF21 (rhFGF21 treatment; lipolysis was assessed. Results. DSS markedly decreased eWAT/body weight ratio and increased serum concentrations of free fatty acid (FFA and glycerol, indicating increased adipose tissue lipolysis. eWAT intracellular lipolytic enzyme expression/activation was significantly increased. These alterations were significantly attenuated in FGF21 KO mice and by circulating FGF21 neutralization. Moreover, DSS treatment markedly increased serum IL-6 and FGF21 levels. IL-6 pretreatment was necessary for the stimulatory effect of FGF21 on adipose lipolysis in 3T3-L1 cells. Conclusions. Our results demonstrate that experimental colitis induces eWAT lipolysis via an IL-6/FGF21-mediated signaling pathway.

  11. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    Directory of Open Access Journals (Sweden)

    Stefan Kippenberger

    Full Text Available Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  12. Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect

    Directory of Open Access Journals (Sweden)

    Nikola Kovářová

    2016-06-01

    Full Text Available This paper describes data related to a research article entitled “Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects” [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1−/− and control (SURF1+/+ mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX, to reversible inhibition of mitochondrial translation in SURF1−/− mouse and SURF1 patient fibroblast cell lines.

  13. Quantitation of the repair of gamma-radiation-induced double-strand DNA breaks in human fibroblasts

    International Nuclear Information System (INIS)

    Woods, W.G.

    1981-01-01

    The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a tsub(1/2) of 10 min for repair of 5000 rad exposure and 39 min for repair of 10000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial fast and a later slow mechanism may be involved. (Auth.)

  14. Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

    Science.gov (United States)

    Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

    2015-07-01

    It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

  15. SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

    Science.gov (United States)

    He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

    2017-01-01

    Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

  16. Dietary moderately oxidized oil induces expression of fibroblast growth factor 21 in the liver of pigs

    Directory of Open Access Journals (Sweden)

    Varady Juliane

    2012-03-01

    Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21, whose expression is induced by peroxisome proliferator-activated receptor α (PPARα, has been recently identified as a novel metabolic regulator which plays a crucial role in glucose homeostasis, lipid metabolism, insulin sensitivity and obesity. Previous studies have shown that administration of oxidized fats leads to an activation of PPARα in the liver. Therefore, the present study investigated the hypothesis that feeding of oxidized fats causes an induction of FGF21 in the liver. Methods Twenty four crossbred pigs were allocated to two groups of 12 pigs each and fed nutritionally adequate diets with either fresh rapeseed oil or oxidized rapeseed oil prepared by heating at a temperature of 175°C for 72 h. Results In pigs fed the oxidized fat mRNA abundance and protein concentrations of FGF21 in liver were significantly increased (P P P Conclusion The present study shows for the first time that administration of an oxidized fat induces the expression of FGF21 in the liver, probably mediated by activation of PPARα. Induction of FGF21 could be involved in several effects observed in animals administered an oxidized fat.

  17. Strawberry-Based Cosmetic Formulations Protect Human Dermal Fibroblasts against UVA-Induced Damage

    Directory of Open Access Journals (Sweden)

    Massimiliano Gasparrini

    2017-06-01

    Full Text Available Extreme exposure of skin to Ultraviolet A (UVA-radiation may induce a dysregulated production of reactive oxygen species (ROS which can interact with cellular biomolecules leading to oxidative stress, inflammation, DNA damage, and alteration of cellular molecular pathways, responsible for skin photoaging, hyperplasia, erythema, and cancer. For these reasons, the use of dietary natural bioactive compounds with remarkable antioxidant activity could be a strategic tool to counteract these UVA-radiation-caused deleterious effects. Thus, the purpose of the present work was to test the efficacy of strawberry (50 μg/mL-based formulations supplemented with Coenzyme Q10 (100 μg/mL and sun protection factor 10 in human dermal fibroblasts irradiated with UVA-radiation. The apoptosis rate, the amount of intracellular reactive oxygen species (ROS production, the expression of proteins involved in antioxidant and inflammatory response, and mitochondrial functionality were evaluated. The results showed that the synergic topical use of strawberry and Coenzyme Q10 provided a significant (p < 0.05 photoprotective effect, reducing cell death and ROS, increasing antioxidant defense, lowering inflammatory markers, and improving mitochondrial functionality. The obtained results suggest the use of strawberry-based formulations as an innovative, natural, and useful tool for the prevention of UVA exposure-induced skin diseases in order to decrease or substitute the amount of synthetic sunscreen agents.

  18. In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide.

    Science.gov (United States)

    Zhao, Guiqing; Yin, Shuo; Liu, Guangpeng; Cen, Lian; Sun, Jian; Zhou, Heng; Liu, Wei; Cui, Lei; Cao, Yilin

    2009-07-01

    This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

  19. Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts

    Directory of Open Access Journals (Sweden)

    Shizuka eMiura

    2014-08-01

    Full Text Available Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.

  20. Infección por Citomegalovarius con compromiso hepático en adultos inmunocompetentes Cytomegalovirus infection with hepatic involvement in immunocompetent adults

    Directory of Open Access Journals (Sweden)

    Claudia Vujacich

    2006-06-01

    presented fever and asthenia, mild to moderate increase of transaminases and serological findings compatible with recent cytomegalovirus infection. We excluded patients with a history of transfusions, drug abuse, immunodeficiencies, preexistent hepatic impairment or serological findings compatible with acute hepatitis A, B and C (HAV, HBV, HCV and Epstein Barr virus (EBV. The laboratory diagnosis of recent cytomegalovirus infection was made by especific IgM detection (ELISA or a significant increase of specific IgG. The most frequent symptoms were fever (85% and asthenia (83%, followed by cephalea (25%, splenomegaly (20%, adenomegalies (22%, pharyngitis (25%, myalgias (25% and hepatomegaly (19%. All the patients showed moderate increase of transaminases and lymphomonocytosis (73/73. In average, ALT was increased by 6 fold and AST by 3.5 fold. The clinical characteristics that differentiate CMV infection from Epstein-Barr infection are the lesser frequency of adenomegalies and pharyngitis in the former. The differential diagnosis of CMV infection with hepatic involvement from acute hepatitis A and B, is based on the absence of jaundice, the lower elevation of transaminases, the intense lymphomonocytosis and the presence of specific IgM against CMV that are characteristic of CMV infection. In conclusion, in previously healthy young adults with fever, intense asthenia, lymphomonocytosis and moderate increase in transaminases levels, cytomegalovirus infection should be investigated.

  1. The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts.

    Science.gov (United States)

    Zhang, Yuan; Tan, Xiaoming; Xue, Lianfang

    2018-01-01

    The α2-adrenoceptor inducer dexmedetomidine protects against acute lung injury (ALI), but the mechanism of this effect is largely unknown. The present study investigated the effect of dexmedetomidine on apoptosis induced by lipopolysaccharide (LPS) and the relationship between this effect and gap junction intercellular communication in human lung fibroblast cell line. Flow cytometry was used to detect apoptosis induced by LPS. Parachute dye coupling assay was used to measure gap junction function, and western blot analysis was used to determine the expression levels of connexin43 (Cx43). The results revealed that exposure of human lung fibroblast cell line to LPS for 24 h increased the apoptosis, and pretreatment of dexmedetomidine and 18α-GA significantly reduced LPS-induced apoptosis. Dexmedetomidine exposure for 1 h inhibited gap junction function mainly via a decrease in Cx43 protein levels in human lung fibroblast cell line. These results demonstrated that the inhibition of gap junction intercellular communication by dexmedetomidine affected the LPS-induced apoptosis through inhibition of gap junction function by reducing Cx43 protein levels. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting ALI. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

    Science.gov (United States)

    Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

    2013-09-01

    Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

  3. Effects of hydroxysafflor yellow A on proliferation and collagen synthesis of rat vascular adventitial fibroblasts induced by angiotensin II.

    Science.gov (United States)

    Yuan, Wendan; Yang, Dongxia; Sun, Xuhong; Liu, Wei; Wang, Liang; Li, Xiaoyan; Man, Xuejing; Fu, Qiang

    2014-01-01

    1) examine the effects of hydroxysafflor yellow A (HSYA) on the proliferation, collagen and cytokine synthesis of vascular adventitial fibroblasts as induced by angiotensin II (Ang II) in normal Sprague-Dawley (SD) rats in vitro, and 2) to assess the effects of HSYA on morphological changes and collagen accumulation of vascular adventitia in spontaneously hypertensive rats (SHR) in vivo. In vitro experiment, vascular adventitial fibroblasts from SD rats were isolated, cultured, and divided into control groups, model groups and HSYA groups. Cell morphology of adventitial fibroblasts was assessed using laser confocal microscopy, while cell proliferation with the MTT assay, and collagen synthesis was determined using hydroxyproline chromatometry. Immunocytochemistry and reverse transcription PCR were used for detecting the expression of TGF-β1, MMP-1, α-SMA and NF-κB in adventitial fibroblasts. In vivo experiment, vascular adventitia proliferation and collagen synthesis were analyzed using hematoxylin-eosin and Sirius staining. Our results showed that: 1) in vitro experiment of SD rats, HSYA inhibited proliferative activity and collagen synthesis of adventitial fibroblasts as induced by Ang II, and the inhibitory effects of HSYA on the increased expression of MMP-1, TGF-β1, α-SMA and NF-κB p65 as induced by Ang II were assessed, and 2) in vivo experiment of SHR, histological analysis displayed fewer pathological changes of vascular adventitia in HSYA treatment groups as compared with no HSYA treatment groups, and MMP-1, TGF-β1, α-SMA and NF-κB p65 expression significantly reduced after HSYA treatment (P adventitia components. This study provides experimental evidence demonstrating that HSYA has the capacity to decrease vascular adventitia proliferation and hyperplasia during vascular remodeling.

  4. Endoglin negatively regulates transforming growth factor beta1-induced profibrotic responses in intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts isolated from strictures in Crohn\\'s disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen. RESULTS: Crohn\\'s stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF. CONCLUSION: Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.

  5. Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

    NARCIS (Netherlands)

    Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

    2015-01-01

    Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their

  6. Pasteurization of breastmilk decreases the rate of postnatally acquired cytomegalovirus infections, but shows a nonsignificant trend to an increased rate of necrotizing enterocolitis in very preterm infants--a preliminary study.

    Science.gov (United States)

    Stock, Katharina; Griesmaier, Elke; Brunner, Barbara; Neubauer, Vera; Kiechl-Kohlendorfer, Ursula; Trawöger, Rudolf

    2015-03-01

    This study assessed whether feeding preterm infants unpasteurized breastmilk (1) decreases the rate of late-onset sepsis and necrotizing enterocolitis and (2) increases the rate of postnatally acquired cytomegalovirus infections. Between January 2008 and July 2013, preterm infants below 32 completed weeks of gestational age admitted to the neonatal intensive care unit of Innsbruck Medical University (Innsbruck, Austria) (n=344) were eligible for the study. Of those, 323 fed breastmilk were retrospectively enrolled in the study. Two groups were formed, with 164 infants being fed unpasteurized and 159 infants being fed pasteurized breastmilk. There was no significant difference in the rate of late-onset sepsis or necrotizing enterocolitis between the unpasteurized and pasteurized breastmilk groups (late-onset sepsis, 15.9% versus 15.1% [p=0.486]; necrotizing enterocolitis, 2.4% versus 4.4% [p=0.254]). The number of infants diagnosed with postnatally acquired cytomegalovirus infection was significantly higher in the unpasteurized group (39.3%) compared with the pasteurized group (4.2%) (p=0.008). Feeding preterm infants unpasteurized breastmilk increases the rate of postnatally acquired cytomegalovirus infections. However, we also demonstrate a nonsignificant trend to a decreased rate of necrotizing enterocolitis in the unpasteurized group, which needs to be confirmed in larger studies.

  7. Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts

    International Nuclear Information System (INIS)

    Tsang, Chi Man; Cheung, Yuk Chun; Lui, Vivian Wai-Yan; Yip, Yim Ling; Zhang, Guitao; Lin, Victor Weitao; Cheung, Kenneth Chat-Pan; Feng, Yibin; Tsao, Sai Wah

    2013-01-01

    Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC

  8. A cigarette component acrolein induces accelerated senescence in human diploid fibroblast IMR-90 cells.

    Science.gov (United States)

    Luo, Cheng; Li, Yan; Yang, Liang; Feng, Zhihui; Li, Yuan; Long, Jiangang; Liu, Jiankang

    2013-10-01

    Cigarette smoking causes various diseases, including lung cancer and cardiovascular disease, and reduces life span, though the mechanisms are not well understood. We hypothesize that smoking may cause cellular mitochondrial dysfunction and oxidative stress, leading to aging acceleration. In the present study, we tested the effects of acrolein, a major representative smoking toxicant, on human lung fibroblast IMR-90 cells with regard to cellular senescence, oxidative stress, and mitochondrial function. The results showed that subacute treatment with low dose of acrolein induces the following events compared to the control cells: cell senescence demonstrated by increases in the activity of β-galactosidase, the higher expression of p53 and p21, decreases in DNA synthesis, Sirt1 expression, and telomere length; oxidative stress occurred as the increases in the production of reactive oxygen species, DNA damage, and protein oxidation; and mitochondrial dysfunction shown as decreases in the mitochondrial membrane potential, mitochondrial biogenesis regulator PGC-1 alpha and mitochondria complex I, II, III, and V. These results suggest that acrolein may accelerate aging through the mechanism of increasing oxidative stress and mitochondrial dysfunction.

  9. The Effects of Amphiregulin Induced MMP-13 Production in Human Osteoarthritis Synovial Fibroblast

    Directory of Open Access Journals (Sweden)

    Yi-Te Chen

    2014-01-01

    Full Text Available Osteoarthritis (OA belongs to a group of degenerative diseases. Synovial inflammation, cartilage abrasion, and subchondral sclerosis are characteristics of OA. Researchers do not fully understand the exact etiology of OA. However, matrix metalloproteinases (MMPs, which are responsible for cartilage matrix degradation, play a pivotal role in the progression of OA. Amphiregulin (AREG binds to the EGF receptor (EGFR and activates downstream proteins. AREG is involved in a variety of pathological processes, such as the development of tumors, inflammatory diseases, and rheumatoid arthritis. However, the relationship between AREG and MMP-13 in OA synovial fibroblasts (SFs remains unclear. We investigated the signaling pathway involved in AREG-induced MMP-13 production in SFs. AREG caused MMP-13 production in a concentration- and time-dependent manner. The results of using pharmacological inhibitors and EGFR siRNA to block EGFR revealed that the EGFR receptor was involved in the AREG-mediated upregulation of MMP-13. AREG-mediated MMP-13 production was attenuated by PI3K and Akt inhibitors. The stimulation of cells by using AREG activated p65 phosphorylation and p65 translocation from the cytosol to the nucleus. Our results provide evidence that AREG acts through the EGFR and activates PI3K, Akt, and finally NF-kappaB on the MMP-13 promoter, thus contributing to cartilage destruction during osteoarthritis.

  10. Distinct cell stress responses induced by ATP restriction in quiescent human fibroblasts

    Directory of Open Access Journals (Sweden)

    Nirupama Yalamanchili

    2016-10-01

    Full Text Available Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

  11. Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1.

    Science.gov (United States)

    Wakao, Junko; Kishida, Tsunao; Fumino, Shigehisa; Kimura, Koseki; Yamamoto, Kenta; Kotani, Shin-Ichiro; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Tajiri, Tatsuro; Mazda, Osam

    2017-06-24

    The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Curcumin induces apoptosis and inhibits prostaglandin E(2) production in synovial fibroblasts of patients with rheumatoid arthritis.

    Science.gov (United States)

    Park, Cheol; Moon, Dong-Oh; Choi, Il-Whan; Choi, Byung Tae; Nam, Taek-Jeong; Rhu, Chung-Ho; Kwon, Taeg Kyu; Lee, Won Ho; Kim, Gi-Young; Choi, Yung Hyun

    2007-09-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by hyperplasia of the synovial fibroblasts, which is partly the result of decreased apoptosis. This study investigated the mechanisms through which curcumin, a polyphenolic compound from the rhizome of Curcuma longa, exerts its anti-proliferative action in the synovial fibroblasts obtained from patients with RA. Exposure of the synovial fibroblasts to curcumin resulted in growth inhibition and the induction of apoptosis, as measured by MTT assay, fluorescent microscopy and Annexin-V-based assay. RT-PCR and immunoblotting showed that treating the cells with curcumin resulted in the down-regulation of anti-apoptotic Bcl-2 and the X-linked inhibitor of the apoptosis protein as well as the up-regulation of pro-apoptotic Bax expression in a concentration-dependent manner. Curcumin-induced apoptosis was also associated with the proteolytic activation of caspase-3 and caspase-9, and the concomitant degradation of poly(ADP-ribose) polymerase protein. Furthermore, curcumin decreased the expression levels of the cyclooxygenase (COX)-2 mRNA and protein without causing significant changes in the COX-1 levels, which was correlated with the inhibition of prostaglandin E(2) synthesis. These results show that curcumin might help identify a new therapeutic pathway against hyperplasia of the synovial fibroblasts in RA.

  13. Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: implications for breast cancer prevention.

    Science.gov (United States)

    Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P

    2013-01-15

    Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with "stemness," more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This "two-compartment" metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert "low-risk" breast cancer patients to "high-risk" status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that

  14. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

    OpenAIRE

    Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

    2014-01-01

    STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductiv...

  15. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-05-01

    Full Text Available We generated human induced pluripotent stem cells (hiPSCs from dermal fibroblasts using a Sendai virus (SeV-based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY. The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  16. Protective effect of curcumin against ultraviolet A irradiation-induced photoaging in human dermal fibroblasts

    Science.gov (United States)

    Liu, Xiaoming; Zhang, Ruizhi; Shi, Haixia; Li, Xiaobo; Li, Yanhong; Taha, Ahmad; Xu, Chunxing

    2018-01-01

    Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in skin, resulting in photoaging. Natural botanicals have gained considerable attention due to their beneficial protection against the harmful effects of UV irradiation. The present study aimed to evaluate the ability of curcumin (Cur) to protect human dermal fibroblasts (HDFs) against ultraviolet A (UVA)-induced photoaging. HDFs were treated with 0–10 µM Cur for 2 h and subsequently exposed to various intensities of UVA irradiation. The cell viability and apoptotic rate of HDFs were investigated by MTT and flow cytometry assays, respectively. The effect of UVA and Cur on the formation of reactive oxygen species (ROS), malondialdehyde levels, which are an indicator of ROS, and the levels/activity of antioxidative defense proteins, including glutathione, superoxide dismutase and catalase, were evaluated using 2′,7′-dichlorofluorescin diacetate and commercial assay kits. Furthermore, western blotting was performed to determine the levels of proteins associated with endoplasmic reticulum (ER) stress, the apoptotic pathway, inflammation and the collagen synthesis pathway. The results demonstrated that Cur reduced the accumulation of ROS and restored the activity of antioxidant defense enzymes, indicating that Cur minimized the damage induced by UVA irradiation in HDFs. Furthermore, western blot analysis demonstrated that Cur may attenuate UVA-induced ER stress, inflammation and apoptotic signaling by downregulating the protein expression of glucose-regulated protein 78, C/EBP-homologous protein, nuclear factor-κB and cleaved caspase-3, while upregulating the expression of Bcl-2. Additionally, it was demonstrated that Cur may regulate collagen metabolism by decreasing the protein expression of matrix metalloproteinase (MMP)-1 and MMP-3, and may promote the repair of cells damaged as a result of UVA irradiation through increasing the protein expression of transforming

  17. The fate of radiation induced giant-nucleated cells of human skin fibroblasts

    Science.gov (United States)

    Almahwasi, A. A.; Jeynes, J. C.; Bradley, D. A.; Regan, P. H.

    2017-11-01

    Radiation-induced giant-nucleated cells (GCs) have been observed to occur within survivors of irradiated cancerous and within healthy cells, both in vivo and in vitro. The expression of such morphological alterations is associated with genomic instability. This study was designed to investigate the fate of GCs induced in a normal human fibroblast cell line (AG1522) after exposure to 0.2, 1 or 2 Gy of X-ray or proton irradiation. The total of 79 individual AG1522 GCs present at 7, 14 or 21 days after each dose point were analysed from fluorescence microscopy images captured over approximately 120 h. The GCs were identified at the beginning of the observation period for each time point post-irradiation and the area of the cell nucleus was measured (μm2) using a cell-recognition MATLAB code. The results demonstrate that the majority of GCs had undergone a prolonged mitotic arrest, which might be an indication of the survival strategy. The live cell microscopy confirms that a giant-nucleated cell formed 14 days after exposure to 0.2 Gy of proton irradiation was divided into two asymmetrical normal-sized cells. These results suggest that a small fraction of GCs can proliferate and form progeny. Some of GCs had disappeared from the microscopy fields. The rate of their loss was decreased as the dose increased but there remains the potential for them to have progeny that could continue to proliferate, ultimately contributing to development of cancer risk. This important method to access delayed effects in normal tissues could act as a potential radioprotective assay for a dose-limiting parameter when applying radiotherapy. These results might have important implications in evaluating risk estimates for patients during radiation therapy treatment.

  18. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  19. Mitochondrial vulnerability and increased susceptibility to nutrient-induced cytotoxicity in fibroblasts from leigh syndrome French canadian patients.

    Directory of Open Access Journals (Sweden)

    Yan Burelle

    Full Text Available Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC, a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol, or modulate fatty acid (L-carnitine or Krebs cycle metabolism (propionate are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise

  20. Improved wound healing in pressure-induced decubitus ulcer with controlled release of basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Jiang Wei; Wang Hailun; Jin Faguang; Yu Chunyan; Chu Dongling; Wang Lin; Lu Xian

    2008-01-01

    The purpose was to evaluate the efficacy of the wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres on promoting healing in pressure-induced decubitus ulcer. In this study, the pressure-induced ulcer in swine was used as a model to demonstrate the hypothesis that controlled release of bFGF has the potential to provide optimal healing milieu for chronic wounds in the repair process. Average size of the microspheres was 14.36 ± 3.56 μm and the network gelatin sponges were characterized with an average pore size of 80-160 μm. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released from the microspheres in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. Pressure-induced ulcer was created at 500 g/cm 2 pressure loaded on swine dorsal skin 12 h daily for 2 consecutive days. After removal of the pressure load, the gelatin sponge containing bFGF gelatin microspheres or bFGF in solution was implanted into the wound. Swine were sacrificed at 7, 14, and 21 days after implantation, and a full-thickness biopsy was taken and stained for histological analysis. It was observed that controlled release of bFGF provided an accelerated recovery in the wound areas. Histological investigations showed that the dressings were biocompatible and had capability of proliferating fibroblasts and inducing neovascularisation. The present study implied the clinical potential of gelatin sponge with bFGF microspheres to promote the healing in pressure-induced decubitus ulcer

  1. Improved wound healing in pressure-induced decubitus ulcer with controlled release of basic fibroblast growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Wei [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Hailun [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Jin Faguang [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)], E-mail: nidewenzhang@163.com; Yu Chunyan [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Chu Dongling [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Lin [Department of Internal Medicine, 316 Hospital of PLA, Beijing 100093 (China); Lu Xian [93942 Unit Hospital of PLA, Xianyang 710012 (China)

    2008-07-14

    The purpose was to evaluate the efficacy of the wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres on promoting healing in pressure-induced decubitus ulcer. In this study, the pressure-induced ulcer in swine was used as a model to demonstrate the hypothesis that controlled release of bFGF has the potential to provide optimal healing milieu for chronic wounds in the repair process. Average size of the microspheres was 14.36 {+-} 3.56 {mu}m and the network gelatin sponges were characterized with an average pore size of 80-160 {mu}m. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released from the microspheres in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. Pressure-induced ulcer was created at 500 g/cm{sup 2} pressure loaded on swine dorsal skin 12 h daily for 2 consecutive days. After removal of the pressure load, the gelatin sponge containing bFGF gelatin microspheres or bFGF in solution was implanted into the wound. Swine were sacrificed at 7, 14, and 21 days after implantation, and a full-thickness biopsy was taken and stained for histological analysis. It was observed that controlled release of bFGF provided an accelerated recovery in the wound areas. Histological investigations showed that the dressings were biocompatible and had capability of proliferating fibroblasts and inducing neovascularisation. The present study implied the clinical potential of gelatin sponge with bFGF microspheres to promote the healing in pressure-induced decubitus ulcer.

  2. Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

    Science.gov (United States)

    Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

    2017-10-01

    The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  3. Proteome alteration induced by hTERT transfection of human fibroblast cells.

    Science.gov (United States)

    Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

    2008-04-17

    Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

  4. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  5. Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

    Science.gov (United States)

    Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming; Wang, Yujiong; He, Yulong

    2017-01-01

    Induced pluripotent stem cells (iPS) represent an important tool to develop disease-modeling assays, drug testing assays and cell-based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 and c-Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder-free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin-coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases. PMID:28393187

  6. Smoking and gingivitis: focus on inducible nitric oxide synthase, nitric oxide and basic fibroblast growth factor.

    Science.gov (United States)

    Özdemir, B; Özmeric, N; Elgün, S; Barış, E

    2016-10-01

    Periodontal disease pathogenesis has been associated with smoking. Gingivitis is a mild and reversible form of periodontal disease and it tends to progress to periodontitis only in susceptible individuals. In the present study, we aimed to examine the impact of smoking on host responses in gingivitis and to evaluate and compare the inducible nitric oxide synthase (iNOS) activity in gingival tissue and NO and basic fibroblast growth factor (bFGF) levels in the gingival crevicular fluid of patients with gingivitis and healthy individuals. Forty-one participants were assigned to the gingivitis-smoker (n = 13), gingivitis (n = 13), healthy-smoker (n = 7) and healthy groups (n = 8). Clinical indices were recorded; gingival biopsy and gingival crevicular fluid samples were obtained from papillary regions. iNOS expression was evaluated by immunohistochemical staining. The immunoreactive cells were semiquantitatively assessed. For the quantitative determination of nitrite and nitrate in gingival crevicular fluid, the NO assay kit was used. The amount of bFGF in gingival crevicular fluid was determined by enzyme-linked immunosorbent assay. The gingivitis-smoker group demonstrated a stronger iNOS expression than the non-smoker gingivitis group. iNOS expression intensity was lower in the non-smoker healthy group compared to that in healthy-smokers. No significant gingival crevicular fluid NO and bFGF level changes were observed between groups. Among patients with gingivitis, a positive correlation was detected between gingival crevicular fluid NO and bFGF levels (r = 0.806, p = 0.001). Our data suggest that smoking has significant effects on iNOS expression but not on gingival crevicular fluid NO or bFGF levels in healthy and patients with gingivitis. However, our results suggest that bFGF might be involved in the regulation of NO production via iNOS. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

    Science.gov (United States)

    Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming Liu; Wang, Yujiong; He, Yulong

    2017-06-01

    Induced pluripotent stem cells (iPS) represent an important tool to develop disease‑modeling assays, drug testing assays and cell‑based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer‑binding transcription factor 4 (Oct4), sex determining region Y‑box 2 (Sox2), Kruppel‑like factor 4 and c‑Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder‑free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin‑coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases.

  8. WNT16B from Ovarian Fibroblasts Induces Differentiation of Regulatory T Cells through β-Catenin Signal in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Cong-Cong Shen

    2014-07-01

    Full Text Available Treatment for cancer can induce a series of secreted factors into the tumor microenvironment, which can affect cancer progression. Wingless-type MMTV (mouse mammary tumor virus integration site 16B (WNT16B is a new member of the WNT family and has been reported to play growth-related roles in previous studies. In this study, we found WNT16B could be expressed and secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. We also demonstrated that fibroblast-derived WNT16B could result in accumulation of β-catenin in dendritic cells and secretion of interleukin-10 (IL-10 and transforming growth factor beta (TGF-β, which contributed to the differentiation of regulatory T cells in a co-culture environment. These results shed light on the roles of WNT16B in immune regulation, especially in regard to cancer treatment.

  9. Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pan

    Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

  10. Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie’s Plaque

    Directory of Open Access Journals (Sweden)

    Dong Hyuk Kang

    2018-05-01

    Full Text Available Purpose: Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs in fibroblasts isolated from plaque tissue of Peyronie’s disease (PD or normal tunica albuginea (TA and to examine the anti-fibrotic effect of small interfering RNA (siRNA-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque. Materials and Methods: For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11 in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL. Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation. Results: The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein. Conclusions: These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

  11. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    International Nuclear Information System (INIS)

    Li, Xiaoou; Liu, Lian; Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan; Wen, Fuqiang

    2014-01-01

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis

  12. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaoou [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Liu, Lian [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Wen, Fuqiang, E-mail: wenfuqiang.scu@gmail.com [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China)

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

  13. Excision of gamma-ray induced thymine lesions by preparations from ataxia telangiectasia fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Remsen, J F; Cerutti, P A [Florida Univ., Gainesville (USA). Inst. of Food and Agricultural Sciences; Florida Univ., Gainesville (USA). Dept. of Biochemistry)

    1977-04-01

    The capacity of whole cell sonicates of skin fibroblasts of normal individuals and patients with the autosomal recessive disease Ataxia telangiectasia (AT) to remove aerobic gamma-ray products of the 5,6-dihydroxydihydrothymine type (tsub(O/sub 2/)sup(..gamma..)) from exogenous DNA substrates was investigated. All four AT strains (AT CRL 1312, AT CRL 1343, AT GM 367, AT 4BI) possessed normal capabilities to excise tsub(O/sub 2/)sup(..gamma..) from irradiated bacteriophage DNA and irradiated chromatin isolated from normal and AT-skin fibroblasts.

  14. Emblica extract prevents cisplatin-induced apoptosis in dermal papilla fibroblasts

    OpenAIRE

    Sudjit Luanpitpong; Varisa Pongrakhananon; Ubonthip Nimmannit; Pithi Chanvorachote

    2008-01-01

    Cisplatin is a widely prescribed anticancer agent that causes hair loss in patients. Since the dermal papilla (DP) fibroblasts are known to be a key mediator in controlling hair growth and loss, understanding the effect and underlying mechanism of cisplatin on these cells may lead to new strategy for hair loss protection in chemotherapy patients. Less is known regarding the effect of cisplatin on DP fibroblasts. We thus treated DP cells with cisplatin (0-250 mmol/L) and found that cisplatin i...

  15. Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

    Science.gov (United States)

    Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

    2013-01-01

    Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

  16. Royal jelly protects against ultraviolet B-induced photoaging in human skin fibroblasts via enhancing collagen production.

    Science.gov (United States)

    Park, Hye Min; Hwang, Eunson; Lee, Kwang Gill; Han, Sang-Mi; Cho, Yunhi; Kim, Sun Yeou

    2011-09-01

    Royal jelly (RJ) is a honeybee product containing proteins, carbohydrates, fats, free amino acids, vitamins, and minerals. As its principal unsaturated fatty acid, RJ contains 10-hydroxy-2-decenoic acid (10-HDA), which may have antitumor and antibacterial activity and a capacity to stimulate collagen production. RJ has attracted interest in various parts of the world for its pharmacological properties. However, the effects of RJ on ultraviolet (UV)-induced photoaging of the skin have not been reported. In this study we measured the 10-HDA content of RJ by high-performance liquid chromatography and tested the effects of RJ on UVB-induced skin photoaging in normal human dermal fibroblasts. The effects of RJ and 10-HDA on UVB-induced photoaging were tested by measuring procollagen type I, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-1 after UVB irradiation. The RJ contained about 0.211% 10-HDA. The UVB-irradiated human skin fibroblasts treated with RJ and 10-HDA had increased procollagen type I and TGF-β1 productions, but the level of MMP-1 was not changed. Thus RJ may potentially protect the skin from UVB-induced photoaging by enhancing collagen production.

  17. Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

    Science.gov (United States)

    Chen, Jian; Lin, Mingyan; Foxe, John J; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M

    2013-01-01

    Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (pdisease-modeling neuropsychiatric disorder and may have some advantages over those derived from F-iPSCs.

  18. Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

    Directory of Open Access Journals (Sweden)

    Julie Carnesecchi

    Full Text Available The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2 addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

  19. Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

    Science.gov (United States)

    Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

    2015-01-01

    The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

  20. The effect of caffeine on X-ray-induced mitotic delay in normal human and ataxia-telangiectasia fibroblasts

    International Nuclear Information System (INIS)

    Zampetti-Bosseler, F.; Scott, D.

    1985-01-01

    The authors previously showed that radiation-sensitive fibroblasts from ataxia-telangiectasia (A-T) patients sustain less G 2 delay after X-irradiation than normal fibroblasts. Caffeine is known to reduce the amount of X-ray-induced delay in various mammalian cell types. It is proposed that A-T cells have an altered chromatin structure, similar to that of caffeine-treated normal cells and that this results in a failure of A-T cells to delay their progression through the cell cycle to allow time for DNA repair. The authors now show that caffeine treatment after X-irradiation reduces G 2 delay in both A-T and normal cells. The authors confirm the results previously obtained on lymphocytes that caffeine potentiates the chromosome-damaging effects of X-rays in both A-T and normal fibroblasts. These and other data suggest that the radiation responses of A-T cells and of caffeine-treated normal cells are caused by different mechanisms. (Auth.)

  1. Evaluating the potential of poly(beta-amino ester nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Bhise NS

    2013-12-01

    Full Text Available Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institut de la Vision, Paris, France*These authors contributed equally to this workBackground: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs from human fibroblasts.Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.Results: 1-(3-aminopropyl-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol polymer (B4S4E7 self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available

  2. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

    Science.gov (United States)

    Yao, Rong; Cao, Yu; He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (ppulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

  3. Increased UV-induced sister-chromatid exchange in cultured fibroblasts of first-degree relatives of melanoma patients

    International Nuclear Information System (INIS)

    Knees-Matzen, S.; Roser, M.; Reimers, U.; Ehlert, U.; Weichenthal, M.; Breitbart, E.W.; Ruediger, H.W.

    1991-01-01

    Cultured fibroblasts of 17 first-degree relatives of familial melanoma patients and six first-degree relatives of cutaneous melanoma (CMM) patients with multiple CMM primaries were tested for in vitro sensitivity to UV light. Fibroblasts of nine familial CMM patients with a known UV-sensitivity and 19 healthy probands served as a control. Sister chromatid exchange (SCE) was used as a parameter to detect UV-induced genotoxic damage. The authors found significantly (p less than 0.001) increased UV-induced SCE levels in familial melanoma patients, as well as in first-degree relatives of familial melanoma patients (p less than 0.001) after UV-A,B irradiation (375 J/m2), compared to the healthy probands without a family history of CMM. A significant (p less than 0.001) increase of UV-induced SCE was also observed in the relatives of CMM patients with multiple CMM primaries. In addition, the spontaneous SCE were significantly increased (p less than 0.05) in familial CMM patients. This study shows that increased UV sensitivity is a familial phenomenon. It is consistent with the concept of a genetic predisposition to CMM, which is based on increased UV sensitivity and may help to define groups with an elevated risk of developing cutaneous malignant melanoma

  4. Ultraviolet-induced formation of micronuclei and sister chromatid exchange in cultured fibroblasts of patients with cutaneous malignant melanoma

    International Nuclear Information System (INIS)

    Roser, M.; Boehm, A.O.; Oldigs, M.; Weichenthal, M.; Reimers, U.; Schmidt-Preuss, U.; Breitbart, E.W.; Ruediger, H.W.

    1989-01-01

    Genetically enhanced sensitivity to ultraviolet (UV) radiation may play an important role in the development of cutaneous malignant melanoma (CMM). This was studied in cultured fibroblasts of 26 CMM patients and controls by micronucleus (MN) test and sister chromatid exchange (SCE) after UV irradiation (375 J/m2). Sister chromatid exchange and MN formation were used as parameters to detect the UV-induced genotoxic damage in the individual cell strains. We found that the UV-induced level of MN was significantly increased in CMM patients (p = 0.0005), being most pronounced in the familial cases (p = 0.0001). Ultraviolet-induced SCE was also elevated in CMM patients (p = 0.001), but there was no difference between familial and nonfamilial cases. The present findings indicate that genetic predisposition contributes to the development of CMM in a subset of CMM patients and may be due to an enhanced susceptibility to UV light

  5. 10-Hydroxy-2-decenoic acid prevents ultraviolet A-induced damage and matrix metalloproteinases expression in human dermal fibroblasts.

    Science.gov (United States)

    Zheng, Jinfen; Lai, Wei; Zhu, Guoxing; Wan, Miaojian; Chen, Jian; Tai, Yan; Lu, Chun

    2013-10-01

    10-Hydroxy-2-decenoic acid (10-HDA) is a major fatty acid component of royal jelly, which has been reported to have a variety of beneficial pharmacological characteristics. However, the effects of 10-HDA on skin photoageing and its potential mechanism of action are unclear. We investigated the protective effects of 10-HDA on ultraviolet (UV) A-induced damage in human dermal fibroblasts (HDFs). We then explored the inhibitory effects of 10-HDA on UVA-induced matrix metalloproteinases (MMPs) expression and elucidated the signalling pathways controlling MMPs inhibition. Primary human dermal fibroblasts were exposed to UVA. Cell proliferation, cellular senescent state and collagen content were analysed using CCK-8, senescence-associated β-galactosidase staining and Sircol collagen assay, respectively. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. The mRNA levels of MMP-1, MMP-3 and type I (α1) collagen were determined by quantitative real-time PCR. Western blot was applied to detect the expression of MMP-1, MMP-3, JNK and p38 MAPK. HDFs treated with 10-HDA were significantly protected from UVA-induced cytotoxicity, ROS, cellular senescence and stimulated collagen production. Moreover, 10-HDA suppressed the UVA-induced expression of MMP-1 and MMP-3 at both the transcriptional and protein levels. Treatment with 10-HDA also reduced the UVA-induced activation of the JNK and p38 MAPK pathways. The data obtained in this study provide evidence that 10-HDA could prevent UVA-induced damage and inhibit MMP-1 and MMP-3 expressions. Therefore, 10-HDA may be a potential agent for the prevention and treatment of skin photoageing. © 2012 The Authors. Journal of the European Academy of Dermatology and Venereology © 2012 European Academy of Dermatology and Venereology.

  6. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

    2016-04-29

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  7. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    International Nuclear Information System (INIS)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li; Li, Xiao-Dong; Hong, Mo-Na; Chen, Qi-Zhi; Han, Wei-Qing; Gao, Ping-Jin

    2016-01-01

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  8. High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

    Science.gov (United States)

    Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

    2016-07-30

    Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

  9. [Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

    Science.gov (United States)

    Song, Chonglin; Zhang, Zhifu; Chen, Xue; Zhang, Yanfeng; Wang, Chunhua; Liu, Keming

    2002-10-01

    To study DNA damage of three kinds of gasoline oxygenates. Single cell gel electrophoresis assay(Comet assay) was used to detect the damage effects of three gasoline oxygenates[methyl tertiary butyl ether(MTBE), ethanol anhydrous(EA) and dimethyl carbonate(DMC)] on DNA in L-929 mice fibroblasts. In certain concentation(37.500-150.000 mg/ml), MTBE could directly cause DNA damage of L-929 mice fibroblasts. There was obvious dose-effect relationship, i.e. when the concentration of MTBE was increased from 9.375 to 150.000 mg/ml, the comet rate also increased from 4% to 85%, and the length of comet tail changed correspondingly. The results of EA and DMC were negative. Under the condition of this experiment(150.000 mg/ml), MTBE could directly cause DNA damage while the effect of EA and DMC on DNA damage was not found.

  10. New isomalabaricane triterpenes from the marine sponge Stelletta globostellata that induce morphological changes in rat fibroblasts.

    Science.gov (United States)

    Oku, N; Matsunaga, S; Wada, S i; Watabe, S; Fusetani, N

    2000-02-01

    Three new isomalabaricane triterpenes, 29-hydroxystelliferin D (2), 3-epi-29-hydroxystelliferin E (3), and 3-epi-29-hydroxystelliferin A (4), were isolated from the marine sponge Stelletta globostellata. Their structures, including absolute stereochemistry, were determined on the basis of spectral data and chemical methods. Rat fibroblasts treated with 0.2 microM of 2-4 exhibited unusual morphological characteristics, followed by death in 5 days.

  11. Hypoxia induces a phenotypic switch of fibroblasts to myofibroblasts through a MMP-2/TIMP mediated pathway: Implications for venous neointimal hyperplasia in hemodialysis access

    Science.gov (United States)

    Misra, Sanjay; Fu, Alex A.; Misra, Khamal D.; Shergill, Uday M.; Leof, Edward B; Mukhopadhyay, Debabrata

    2010-01-01

    Purpose Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts which have become myofibroblasts (α-smooth muscle actin positive cells) and migrate to the neointima. There is increased expression of hypoxia inducible factor-1 alpha (HIF-1α in venous neointimal hyperplasia formation in experimental animal model and clinical samples. We hypothesized that under hypoxic stimulus (HIF-1α fibroblasts will convert to myofibroblasts through a matrix metalloproteinase-2 (MMP-2) mediated pathway. Materials and methods Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1α, α-smooth muscle actin, MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for α-smooth muscle actin, collagen, and fibronectin was performed. Results At all time points, there was significantly increased expression of HIF-1α in the hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was significantly increased expression α-smooth muscle actin at all time points which peaked by 48 hours in hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) by 48-72 hours by hypoxic fibroblasts (P<0.05). By 72 hours, there was significant increase in TIMP-2 expression (P<0.05). Immunohistochemical analysis demonstrated increased expression for α-smooth muscle actin, collagen, and fibronectin as the length of hypoxia increased. Conclusions Under hypoxia, fibroblasts will convert to myofibroblasts through a MMP-2 mediated pathway which may provide insight into the mechanism of venous neointimal hyperplasia. PMID:20434368

  12. Molecular alterations of tropoelastin and proteoglycans induced by tobacco smoke extracts and ultraviolet A in cultured skin fibroblasts

    International Nuclear Information System (INIS)

    Yin, Lei; Morita, Akimichi; Tsuji, Takuo

    2002-01-01

    Functional integrity of normal skin is dependent on the balance between the biosynthesis and degradation of extracellular matrix, primarily composed of collagen, elastin and proteoglycans. In our previous studies, we found that tobacco smoke extracts decreased expressions of type I and III procollagen and induced matrix metalloproteinase-1 (MMP-1) and MMP-3 in the cultured skin fibroblasts. We here further investigated the effects of tobacco smoke extracts or ultraviolet A (UVA) treatments on the expression of tropoelastin (soluble elastin protein), and versican and decorin (proteoglycans) in cultured skin fibroblasts. The mRNA of tropoelastin increased by tobacco smoke extracts or UVA irradiation. Versican was markedly shown to decrease after these treatments by using western blotting and the mRNA of versican V0 also significantly decreased. UVA treatment did not show remarkable change in decorin protein, but resulted in marked decrease of decorin D1 mRNA. In contrast to UVA irradiation, the treatments of tobacco smoke extracts resulted in significant increase in decorin, while mRNA of decorin D1 decreased as compared to the control. MMP-7 increased after the treatment of tobacco smoke extracts or UVA. These results indicated that common molecular features might underlie the skin premature aging induced by tobacco smoke extracts and UVA, including abnormal regulation of extracellular matrix deposition through elevated MMPs, reduced collagen production, abnormal tropoelastin accumulation, and altered proteoglycans. (author)

  13. Molecular alterations of tropoelastin and proteoglycans induced by tobacco smoke extracts and ultraviolet A in cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Lei; Morita, Akimichi; Tsuji, Takuo [Nagoya City Univ. (Japan). Medical School

    2002-02-01

    Functional integrity of normal skin is dependent on the balance between the biosynthesis and degradation of extracellular matrix, primarily composed of collagen, elastin and proteoglycans. In our previous studies, we found that tobacco smoke extracts decreased expressions of type I and III procollagen and induced matrix metalloproteinase-1 (MMP-1) and MMP-3 in the cultured skin fibroblasts. We here further investigated the effects of tobacco smoke extracts or ultraviolet A (UVA) treatments on the expression of tropoelastin (soluble elastin protein), and versican and decorin (proteoglycans) in cultured skin fibroblasts. The mRNA of tropoelastin increased by tobacco smoke extracts or UVA irradiation. Versican was markedly shown to decrease after these treatments by using western blotting and the mRNA of versican V0 also significantly decreased. UVA treatment did not show remarkable change in decorin protein, but resulted in marked decrease of decorin D1 mRNA. In contrast to UVA irradiation, the treatments of tobacco smoke extracts resulted in significant increase in decorin, while mRNA of decorin D1 decreased as compared to the control. MMP-7 increased after the treatment of tobacco smoke extracts or UVA. These results indicated that common molecular features might underlie the skin premature aging induced by tobacco smoke extracts and UVA, including abnormal regulation of extracellular matrix deposition through elevated MMPs, reduced collagen production, abnormal tropoelastin accumulation, and altered proteoglycans. (author)

  14. Characterization of ultraviolet light-induced diphtheria toxin-resistant mutations in normal and Xeroderma pigmentosum human fibroblasts

    International Nuclear Information System (INIS)

    Glover, T.W.

    1979-01-01

    Quantitative mutagenesis studies in human cells have been severely limited by the lack of reliable genetic markers. Experiments were therefore performed to develop and characterize a better quantitative mutation assay for human cells. The uv-induction of diphtheria toxin resistant (DT/sup r/) mutations in normal and excision repair defective xeroderma pigmentosum (XP) fibroblasts has been quantitatively characterized. A concentration of diphtheria toxin to use in the selection of resistant mutants was determined whereby DT/sup r/ cells are cross-resistant to Pseudomonas aeurginosa exotoxin A, indicating mutants have altered elongation factor-2 (EF-2) which is not susceptible to ADP-ribosylation by either toxin. Results of this study indicate that XP fibroblasts have higher uv-induced mutation frequencies per unit uv-dose but similar frequencies per unit survival compared to normal cells as measured using a new genetic marker for quantitative mutagenesis. Furthermore, these results support a prediction of the mutation theory of cancer, namely, that cells from individuals with certain human syndromes that predispose the individual to cancer will have higher induced mutation frequencies than cells from non-susceptible individuals. This newly characterized genetic marker should be useful in quantitative mutagenesis studies in human cells

  15. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Yukihiro Nishikawa

    Full Text Available Withaferin A (WA, a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD. WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L. Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

  16. Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.

    Science.gov (United States)

    Kim, Seong Hun; Kim, Kook Hwan; Kim, Hyoung-Kyu; Kim, Mi-Jeong; Back, Sung Hoon; Konishi, Morichika; Itoh, Nobuyuki; Lee, Myung-Shik

    2015-04-01

    Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-diabetic and anti-obesity activity. FGF21 expression is increased in patients with and mouse models of obesity or nonalcoholic fatty liver disease (NAFLD). However, the functional role and molecular mechanism of FGF21 induction in obesity or NAFLD are not clear. As endoplasmic reticulum (ER) stress is triggered in obesity and NAFLD, we investigated whether ER stress affects FGF21 expression or whether FGF21 induction acts as a mechanism of the unfolded protein response (UPR) adaptation to ER stress induced by chemical stressors or obesity. Hepatocytes or mouse embryonic fibroblasts deficient in UPR signalling pathways and liver-specific eIF2α mutant mice were employed to investigate the in vitro and in vivo effects of ER stress on FGF21 expression, respectively. The in vivo importance of FGF21 induction by ER stress and obesity was determined using inducible Fgf21-transgenic mice and Fgf21-null mice with or without leptin deficiency. We found that ER stressors induced FGF21 expression, which was dependent on a PKR-like ER kinase-eukaryotic translation factor 2α-activating transcription factor 4 pathway both in vitro and in vivo. Fgf21-null mice exhibited increased expression of ER stress marker genes and augmented hepatic lipid accumulation after tunicamycin treatment. However, these changes were attenuated in inducible Fgf21-transgenic mice. We also observed that Fgf21-null mice with leptin deficiency displayed increased hepatic ER stress response and liver injury, accompanied by deteriorated metabolic variables. Our results suggest that FGF21 plays an important role in the adaptive response to ER stress- or obesity-induced hepatic metabolic stress.

  17. The Effects of Age and Latent Cytomegalovirus Infection on NK-Cell Phenotype and Exercise Responsiveness in Man

    Directory of Open Access Journals (Sweden)

    Austin B. Bigley

    2015-01-01

    Full Text Available The redeployment of NK-cells in response to an acute bout of exercise is thought to be an integral component of the “fight-or-flight” response, preparing the body for potential injury or infection. We showed previously that CMV seropositivity impairs the redeployment of NK-cells with exercise in the young. In the current study, we examined the effect of aging on the redeployment of NK-cells with exercise in the context of CMV. We show here that CMV blunts the exercise-induced redeployment of NK-cells in both younger (23–39 yrs and older (50–64 yrs subjects with older CMVneg subjects showing the largest postexercise mobilization and 1 h postexercise egress of NK-cells. The blunted exercise response in CMVpos individuals was associated with a decreased relative redeployment of the CD158a+ and CD57+ NK-cell subsets in younger and older individuals. In addition, we show that aging is associated with a CMV-independent increase in the proportion of NK-cells expressing the terminal differentiation marker CD57, while CMV is associated with an age-dependent decrease in the proportion of NK-cells expressing the inhibitory receptors KLRG1 (in the younger group and CD158a (in the older group. Collectively, these data suggest that CMV may decrease NK-cell mediated immunosurveillance after exercise in both younger and older individuals.

  18. Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

    Directory of Open Access Journals (Sweden)

    Felix R Stahl

    Full Text Available Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8(+ T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming "nodular inflammatory foci" (NIF in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

  19. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Hiromi Murase

    2013-01-01

    Full Text Available Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB. Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8 cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS- sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

  20. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Alsner, Jan; Rødningen, Olaug K.; Overgaard, Jens

    2007-01-01

    BACKGROUND AND PURPOSE: Differentially gene expression between patients with either very low or very high risk of radiation-induced fibrosis (RIF) in patient-derived fibroblasts after irradiation has previously been reported. In the present study, we are investigating the robustness of radiation...... and changes in radiation-induced gene expression in fibroblasts. MATERIAL AND METHODS: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3x3.5Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy....... RESULTS: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk...

  1. Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

    Directory of Open Access Journals (Sweden)

    Ferry Sandra

    2017-09-01

    Full Text Available Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS.  Human umbilical cord blood (hUCB has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS could be potential and were investigated in current study. Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay. Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000. Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000. Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast. Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

  2. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Salahuddin, E-mail: Salah.Ahmed@utoledo.edu [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  3. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    International Nuclear Information System (INIS)

    Mizoshiri, N.; Kishida, T.; Yamamoto, K.; Shirai, T.; Terauchi, R.; Tsuchida, S.; Mori, Y.; Ejima, A.; Sato, Y.; Arai, Y.; Fujiwara, H.; Yamamoto, T.; Kanamura, N.; Mazda, O.; Kubo, T.

    2015-01-01

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  4. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  5. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    Science.gov (United States)

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  6. Imaging Features that Discriminate between Foci Induced by High- and Low-LET Radiation in Human Fibroblasts

    International Nuclear Information System (INIS)

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano, Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-01-01

    In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90 percent compared to 60 percent after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities

  7. Imaging Features that Discriminate between Foci Induced by High-and Low-LET Radiation in Human Fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano,Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-10-08

    In this study, we investigated the formation ofradiation-induced foci in normal human fibroblasts exposed to X rays or130 keV/mum nitrogen ions using antibodies to phosphorylated proteinkinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantifythe immunofluorescence of radiation-induced foci. The size ofradiation-induced foci increased for both proteins over a 2-h periodafter nitrogen-ion irradiation, while the size of radiation-induced focidid not change after exposure to low-LET radiation. The number ofradiation-induced ATMp foci showed a more rapid rise and greaterfrequency after X-ray exposure and was resolved more rapidly such thatthe frequency of radiation-induced foci decreased by 90 percent comparedto 60 percent after exposure to high-LET radiation 2 h after 30 cGy. Incontrast, the kinetics of radiation-induced gamma-H2AX focus formationwas similar for high- and low-LET radiation in that it reached a plateauearly and remained constant for up to 2 h. High-resolution 3D images ofradiation-induced gamma-H2AX foci and dosimetry computation suggest thatmultiple double-strand breaks from nitrogen ions are encompassed withinlarge nuclear domains of 4.4 Mbp. Our work shows that the size andfrequency of radiation-induced foci vary as a function of radiationquality, dose, time and protein target. Thus, even though double-strandbreaks and radiation-induced foci are correlated, the dynamic nature ofboth contradicts their accepted equivalence for low doses of differentradiation qualities.

  8. Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

    Science.gov (United States)

    Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

    2013-01-01

    Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This

  9. Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts

    International Nuclear Information System (INIS)

    Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.

    1987-01-01

    Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA

  10. Near-ultraviolet radiation-induced lipid peroxidation and membrane effects in Escherichia coli and human skin fibroblasts

    International Nuclear Information System (INIS)

    Chamberlain, J.

    1987-01-01

    The first part of this thesis examines the response of an unsaturated fatty acid auxotroph, Escherichia coli K1060 to broad-band near-UV radiation. Sensitivity, lipid peroxidation and leakage of rubidium from irradiated cells were found to increase with increasing unsaturation of membrane fatty acids. The involvement of singlet oxygen was implicated by an increase in sensitivity, lipid peroxidation and leakage of rubidium following irradiation in deuterium oxide. Some factors influencing survival following irradiation were investigated, where lower growth rates were shown to enhance survival. In the second part, the study was extended to human fibroblasts where a normal human skin fibroblast strain, GM730 and a strain derived from an actinic reticuloid patient, AR6LO, are compared. Lipid peroxidation was measured in both cell lines following broad-band near-UV irradiation. Membrane activity, as assessed by the pinocytic uptake of 14 C-sucrose and its subsequent release from the cell, was measured. Near-UV irradiation was found to increase such activity in both strains. Vitamin E and Trolox-C were found to decrease this response in AR6LO but not GM730 cells. The final part consists of preliminary investigations into the near-UV induced peroxidation of fatty acids and liposomes, and the subsequent increase in the level of hydroperoxides in the hours following irradiation. (author)

  11. Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

    Science.gov (United States)

    Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy

    2015-01-01

    Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. PMID:26100252

  12. Breast cancer cells induce stromal fibroblasts to secrete ADAMTS1 for cancer invasion through an epigenetic change.

    Directory of Open Access Journals (Sweden)

    Shiaw-Wei Tyan

    Full Text Available Microenvironment plays an important role in cancer development. We have reported that the cancer-associated stromal cells exhibit phenotypic and functional changes compared to stromal cells neighboring to normal tissues. However, the molecular mechanisms as well as the maintenance of these changes remain elusive. Here we showed that through co-culture with breast cancer cells for at least three to four passages, breast normal tissue-associated fibroblasts (NAFs gained persistent activity for promoting cancer cell invasion, partly via up-regulating ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1. Furthermore, we demonstrated that the DNA methylation pattern in the ADAMTS1 promoter has no alteration. Instead, the loss of EZH2 binding to the ADAMTS1 promoter and the resulting decrease of promoter-associated histone H3K27 methylation may account for the up-regulation of ADAMTS1. Importantly, the lack of EZH2 binding and the H3K27 methylation on the ADAMTS1 promoter were sustained in cancer cell-precocultured NAFs after removal of cancer cells. These results suggest that cancer cells are capable of inducing stromal fibroblasts to secrete ADAMTS1 persistently for their invasion and the effect is epigenetically inheritable.

  13. Mitochondrial reactive oxygen species mediate the lipopolysaccharide-induced pro-inflammatory response in human gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xue; Wang, Xiaoxuan [Department of Periodontology, Peking University School and Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Zheng, Ming, E-mail: zhengm@bjmu.edu.cn [Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing 100191 (China); Luan, Qing Xian, E-mail: kqluanqx@126.com [Department of Periodontology, Peking University School and Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China)

    2016-09-10

    Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone. Mitochondrial reactive oxygen species (mtROS) have been proposed to regulate the activation of the inflammatory response by the innate immune system. However, the role of mtROS in modulating the response of human gingival fibroblasts (HGFs) to immune stimulation by lipopolysaccharides (LPS) has yet to be fully elucidated. Here, we showed that LPS from Porphyromonas gingivalis stimulated HGFs to increase mtROS production, which could be inhibited by treatment with a mitochondrial-targeted exogenous antioxidant (mito-TEMPO) or transfection with manganese superoxide dismutase (MnSOD). A time-course study revealed that an increase in the concentration of mtROS preceded the expression of inflammatory cytokines in HGFs. Mito-TEMPO treatment or MnSOD transfection also significantly prevented the LPS-induced increase of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Furthermore, suppressing LPS-induced mtROS generation inhibited the activation of p38, c-Jun N-terminal kinase, and inhibitor of nuclear factor-κB kinase, as well as the nuclear localization of nuclear factor-κB. These results demonstrate that mtROS generation is a key signaling event in the LPS-induced pro-inflammatory response of HGFs. - Highlights: • Inflammation is thought to promote pathogenic changes in periodontitis. • We investigated mtROS as a regulator of inflammation in gingival fibroblasts. • Targeted antioxidants were used to inhibit mtROS production after LPS challenge. • Inhibiting mtROS generation suppressed the secretion of pro-inflammatory cytokines. • JNK, p38, IKK, and NF-κB were shown to act as transducers of mtROS signaling.

  14. Exposure to titanium dioxide and other metallic oxide nanoparticles induces cytotoxicity on human neural cells and fibroblasts

    Directory of Open Access Journals (Sweden)

    James C K Lai

    2008-12-01

    Full Text Available James C K Lai1, Maria B Lai1, Sirisha Jandhyam1, Vikas V Dukhande1, Alok Bhushan1, Christopher K Daniels1, Solomon W Leung21Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, and Biomedical Research Institute; 2Department of Civil and Environmental Engineering, College of Engineering and Biomedical Research Institute, Idaho State University, Pocatello, ID, USAAbstract: The use of titanium dioxide (TiO2 in various industrial applications (eg, production of paper, plastics, cosmetics, and paints has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO2 nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO2 micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO nanoparticles were the most effective, TiO2 nanoparticles the second most effective, and magnesium oxide (MgO nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO2 micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.Keywords: cytotoxicity of titanium dioxide micro- and nanoparticles, cytotoxicity of zinc oxide and magnesium oxide nanoparticles, human neural cells

  15. Mitochondrial reactive oxygen species mediate the lipopolysaccharide-induced pro-inflammatory response in human gingival fibroblasts

    International Nuclear Information System (INIS)

    Li, Xue; Wang, Xiaoxuan; Zheng, Ming; Luan, Qing Xian

    2016-01-01

    Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone. Mitochondrial reactive oxygen species (mtROS) have been proposed to regulate the activation of the inflammatory response by the innate immune system. However, the role of mtROS in modulating the response of human gingival fibroblasts (HGFs) to immune stimulation by lipopolysaccharides (LPS) has yet to be fully elucidated. Here, we showed that LPS from Porphyromonas gingivalis stimulated HGFs to increase mtROS production, which could be inhibited by treatment with a mitochondrial-targeted exogenous antioxidant (mito-TEMPO) or transfection with manganese superoxide dismutase (MnSOD). A time-course study revealed that an increase in the concentration of mtROS preceded the expression of inflammatory cytokines in HGFs. Mito-TEMPO treatment or MnSOD transfection also significantly prevented the LPS-induced increase of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Furthermore, suppressing LPS-induced mtROS generation inhibited the activation of p38, c-Jun N-terminal kinase, and inhibitor of nuclear factor-κB kinase, as well as the nuclear localization of nuclear factor-κB. These results demonstrate that mtROS generation is a key signaling event in the LPS-induced pro-inflammatory response of HGFs. - Highlights: • Inflammation is thought to promote pathogenic changes in periodontitis. • We investigated mtROS as a regulator of inflammation in gingival fibroblasts. • Targeted antioxidants were used to inhibit mtROS production after LPS challenge. • Inhibiting mtROS generation suppressed the secretion of pro-inflammatory cytokines. • JNK, p38, IKK, and NF-κB were shown to act as transducers of mtROS signaling.

  16. Stretched inverse opal colloid crystal substrates-induced orientation of fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Y C; Tang, Z M; Feng, Z Q; Xie, Z Y; Gu, Z Z, E-mail: gu@seu.edu.c [State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096 (China)

    2010-06-01

    Recently, there has been increasing interest in studying the interaction between mammalian cells and nanometer-sized structures. However, the effect of nanostructures on cell behavior, such as cell morphology and alignment, is still largely unknown. Inverse opal colloid crystal substrates, which can be stretched to produce nano-scale pore structures of different degrees of orientation, serve as a convenient model system to study the effect of nanotopography on cell morphology and cell alignment. In this work, we fabricated inverse opal colloidal crystal films that were either unstretched or stretched to three, four or six times their original length, producing pore structures of increasing degree of orientation. Human dermal fibroblast-fetal (HDF-f) cells were seeded and cultured on these four types of substrates. The results from fluorescence microscopy and scanning electron microscopy indicated that cells showed the highest degree of alignment when cultured on inverse opal colloid crystal films that were stretched the most (six times original length). The results also demonstrated that the orientation of nanostructures could affect both the morphology and growth direction of fibroblasts. The ability to control the direction of cell growth through the engineering of nanostructures could have important applications in tissue engineering, especially for tissues with anisotropic structures, such as cardiac muscle, blood vessel, tendon and ligament.

  17. Stretched inverse opal colloid crystal substrates-induced orientation of fibroblast

    International Nuclear Information System (INIS)

    Wang, Y C; Tang, Z M; Feng, Z Q; Xie, Z Y; Gu, Z Z

    2010-01-01

    Recently, there has been increasing interest in studying the interaction between mammalian cells and nanometer-sized structures. However, the effect of nanostructures on cell behavior, such as cell morphology and alignment, is still largely unknown. Inverse opal colloid crystal substrates, which can be stretched to produce nano-scale pore structures of different degrees of orientation, serve as a convenient model system to study the effect of nanotopography on cell morphology and cell alignment. In this work, we fabricated inverse opal colloidal crystal films that were either unstretched or stretched to three, four or six times their original length, producing pore structures of increasing degree of orientation. Human dermal fibroblast-fetal (HDF-f) cells were seeded and cultured on these four types of substrates. The results from fluorescence microscopy and scanning electron microscopy indicated that cells showed the highest degree of alignment when cultured on inverse opal colloid crystal films that were stretched the most (six times original length). The results also demonstrated that the orientation of nanostructures could affect both the morphology and growth direction of fibroblasts. The ability to control the direction of cell growth through the engineering of nanostructures could have important applications in tissue engineering, especially for tissues with anisotropic structures, such as cardiac muscle, blood vessel, tendon and ligament.

  18. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

    2008-01-01

    in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar......BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...

  19. Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x Skin fibroblast human cell hybrids

    International Nuclear Information System (INIS)

    Mendonca, M.S.; Redpath, J.L.; Fasching, C.L.

    1995-01-01

    The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to γ radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several γ-ray-induced IAP-expression mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice. Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. 49 refs., 3 figs., 2 tabs

  20. Neurological Consequences of Cytomegalovirus Infection

    Science.gov (United States)

    ... that causes cold sores (herpes simplex virus), infectious mononucleosis (Epstein-Barr virus), and chickenpox/shingles (varicella zoster ... that causes cold sores (herpes simplex virus), infectious mononucleosis (Epstein-Barr virus), and chickenpox/shingles (varicella zoster ...

  1. GWAS, Cytomegalovirus Infection, and Schizophrenia

    DEFF Research Database (Denmark)

    Grove, Jakob; Børglum, Anders; Pearce, Brad D

    2014-01-01

    In recent years, good progress has been made in uncovering the genetic underpinnings of schizophrenia. Even so, as a polygenic disorder, schizophrenia has a complex etiology that is far from understood. Meanwhile, data are being collected enabling the study of interactions between genes...... and the environment. A confluence of data from genetic and environmental exposure studies point to the role of infections and immunity in the pathophysiology of schizophrenia. In a recent study by Børglum et al., a single nucleotide polymorphism (SNP) in the gene CTNNA3 was identified that may provide clues to gene......-environment interactions. The carriers of the minor allele for the SNP had a fivefold risk of later developing schizophrenia if their mothers were CMV positive, while the children not carrying the allele had no excess risk from maternal CMV. In the current paper, we summarize recent advances to clarify a possible...

  2. Congenital and perinatal cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Chun Soo Kim

    2010-01-01

    Full Text Available Cytomegalovirus (CMV is currently the most common agent of congenital infection and the leading infectious cause of brain damage and hearing loss in children. Symptomatic congenital CMV infections usually result from maternal primary infection during early pregnancy. One half of symptomatic infants have cytomegalic inclusion disease (CID, which is characterized by involvement of multiple organs, in particular, the reticuloendothelial and central nervous system (CNS. Moreover, such involvement may or may not include ocular and auditory damage. Approximately 90% of infants with congenital infection are asymptomatic at birth. Preterm infants with perinatal CMV infection can have symptomatic diseases such as pneumonia, hepatitis, and thrombocytopenia. Microcephaly and abnormal neuroradiologic imaging are associated with a poor prognosis. Hearing loss may occur in both symptomatic and asymptomatic infants with congenital infection and may progress through childhood. Congenital infection is defined by the isolation of CMV from infants within the first 3 weeks of life. Ganciclovir therapy can be considered for infants with symptomatic congenital CMV infection involving the CNS. Pregnant women of seronegative state should be counseled on the importance of good hand washing and other control measures to prevent CMV infection. Heat treatment of infected breast milk at 72?#608;for 5 seconds can eliminate CMV completely.

  3. Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts.

    Science.gov (United States)

    Min, Jie; Li, Bingshu; Liu, Cheng; Guo, Wenjun; Hong, Shasha; Tang, Jianming; Hong, Li

    2017-05-01

    Pelvic organ prolapse (POP) is a global health problem that may seriously impact the quality of life of the sufferer. The present study aimed to investigate the potential mechanisms underlying alterations in extracellular matrix (ECM) metabolism in the pathogenesis of POP, by investigating the expression of ECM components in human parametrial ligament fibroblasts (hPLFs) subject to various mechanical strain loads. Fibroblasts derived from parametrial ligaments were cultured from patients with POP and without malignant tumors, who underwent vaginal hysterectomy surgery. Fibroblasts at generations 3‑6 of exponential phase cells were selected, and a four‑point bending device was used for 0, 1,333 or 5,333 µ mechanical loading of cells at 0.5 Hz for 4 h. mRNA and protein expression levels of collagen type I α 1 chain (COL1A1), collagen type III α 1 chain (COL3A1), elastin, matrix metalloproteinase (MMP) ‑2 and ‑9, and transforming growth factor (TGF)‑β1 were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Under increased mechanical strain (5,333 µ), mRNA and protein expression levels of COL1A1, COL3A1 elastin and TGF‑β1 decreased, particularly COL1A1; however, mRNA and protein expression levels of MMP‑2 and ‑9 were significantly increased, compared with the control group (0 µ strain). Following 1,333 µ mechanical strain, mRNA and protein expression levels of COL1A1, COL3A1 elastin and MMP‑2 increased, and MMP‑9 decreased, whereas no significant differences were observed in TGF‑β1 mRNA and protein expression levels. In conclusion, ECM alterations may be involved in pathogenesis of POP, with decreased synthesis and increased degradation of collagen and elastin. Furthermore, the TGF‑β1 signaling pathway may serve an important role in this process and thus may supply a new target and strategy for understanding the etiology and therapy of POP.

  4. Molecular mechanisms of UVB-induced senescence of dermal fibroblasts and its relevance for photoaging of the human skin.

    Science.gov (United States)

    Cavinato, Maria; Jansen-Dürr, Pidder

    2017-08-01

    Due to its ability to cross the epidermis and reach the upper dermis where it causes cumulative DNA damage and increased oxidative stress, UVB is considered the most harmful component of sunlight to the skin. The consequences of chronic exposition to UVB are related to photoaging and photocarcinogenesis. There are limitations to the study of human skin aging and for this reason the use of models is required. Human dermal fibroblasts submitted to mild and repeated doses of UVB are considered a versatile model to study UVB effects in the process of skin photoaging, which depends on the accumulation of senescent cells, in particular in the dermis. Here we provide updated information about the current model of UVB-induced senescence with special emphasis on the process of protein quality control. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Neuroligin-1 induces neurite outgrowth through interaction with neurexin-1ß and activation of fibroblast growth factor receptor-1

    DEFF Research Database (Denmark)

    Gjørlund, Michelle D; Nielsen, Janne; Pankratova, Stanislava

    2012-01-01

    Neurexin-1 (NRXN1) and neuroligin-1 (NLGN1) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses and mediate signaling across the synapse, which modulates synaptic activity and determines the properties of neuronal networks. Defects in the genes encoding NLGN1...... have been linked to cognitive diseases such as autism. The roles of both NRXN1 and NLGN1 during synaptogenesis have been studied extensively, but little is known about the role of these molecules in neuritogenesis, which eventually results in neuronal circuitry formation. The present study investigated...... the neuritogenic effect of NLGN1 in cultures of hippocampal neurons. Our results show that NLGN1, both in soluble and membrane-bound forms, induces neurite outgrowth that depends on the interaction with NRXN1ß and on activation of fibroblast growth factor receptor-1. In addition, we demonstrate that a synthetic...

  6. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-10-01

    Full Text Available We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1 from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4(q21;q25 that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY. These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  7. Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

    International Nuclear Information System (INIS)

    Smith, P.J.; Paterson, M.C.

    1980-01-01

    The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells was accompanied by a lack of inhibition of DNA synthesis by either γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic-type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative

  8. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Yingying; Chen, Xi; Yu, Dehai [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Tao [Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Cui, Jiuwei; Wang, Guanjun [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Hu, Ji-Fan, E-mail: jifan@stanford.edu [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Wei, E-mail: jdyylw@163.com [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China)

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  9. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    International Nuclear Information System (INIS)

    Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

    2009-01-01

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2 -/- mouse embryonic fibroblasts (MEFs) while Akt1 -/- MEFs show cell cycle arrest. Here, we find that Akt1 -/- MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1 -/- MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1 -/- MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1 -/- MEFs, suggesting that UV light induces premature senescence in Akt1 -/- MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  10. Differential gene expression in primary fibroblasts induced by proton and cobalt-60 beam irradiation

    DEFF Research Database (Denmark)

    Nielsen, Steffen; Bassler, Niels; Grzanka, Leszek

    2017-01-01

    profile: entrance, mid-SOBP and at the SOBP distal edge. Dose was delivered in three fractions × 3.5 Gy(RBE) (RBE 1.1). Cobalt-60 (Co-60) irradiation was used as reference. Real-time qPCR was performed to determine gene expression levels for 17 genes associated with inflammation response, fibrosis...... and angiogenesis. RESULTS: Differences in median gene expression levels were observed for multiple genes such as IL6, IL8 and CXCL12. Median IL6 expression was 30%, 24% and 47% lower in entrance, mid-SOBP and SOBP distal edge groups than in Co-60 irradiated cells. No genes were found to be oppositely regulated...... fibroblast cultures. Inflammatory factors were generally less extensively upregulated by proton irradiation compared with Co-60 photon irradiation. These effects may possibly influence the development of normal tissue damage in patients treated with proton beam therapy....

  11. Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

    Science.gov (United States)

    Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

    2015-12-01

    We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

    Directory of Open Access Journals (Sweden)

    Ribeiro-Resende Victor

    2012-07-01

    Full Text Available Abstract Background Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC, satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2 is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS. Results Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL region of the lumbar spinal cord (LSC in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. Conclusion We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.

  13. Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Whittam, Alexander J; Sorkin, Michael; Hu, Michael S; Walmsley, Graham G; Baker, Hutton; Fischer, Lauren H; Januszyk, Michael; Wong, Victor W; Gurtner, Geoffrey C

    2015-11-01

    Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model. The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p wound closure and vascularity (p wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

  14. Solar-simulated radiation and heat treatment induced metalloproteinase-1 expression in cultured dermal fibroblasts via distinct pathways: implications on reduction of sun-associated aging.

    Science.gov (United States)

    Lan, Cheng-Che E; Wu, Ching-Shang; Yu, Hsin-Su

    2013-12-01

    Sun exposure is an important environmental factor affecting human beings. Most knowledge regarding solar aging focused on light radiation (photoaging), and little emphasis has been placed on heat, a factor that is also closely associated with sun exposure. This study was launched to evaluate the effects of simulated solar radiation (SSR) and environmental heat on skin fibroblasts in terms of dermal aging. Cultured human dermal fibroblasts were treated with moderate amount of SSR (200J/cm(2)) and heat (+2°C). The metalloproteinase-1 (MMP-1) expression was used as a surrogate marker for dermal aging and the involved regulatory mechanisms were explored. Both treatment conditions did not affect viability but significantly increased the expressions of MMP-1. In parallel, both treatments increased the intracellular levels of reactive oxygen species (ROS), but the increase induced by SSR is much greater than heat. In contrast, transient receptor potential vanilloid 1 (TRPV-1), the sensor of environmental heat, was upregulated by heat but not SSR treatment. Pretreating fibroblasts with antioxidant abrogated the SSR-induced MMP-1 but has limited effect on heat-induced MMP-1. On the other hand, TRPV-1 antagonist pretreatment reduced heat-induced MMP-1 in fibroblasts but not their SSR-treated counterparts. Both SSR and heat induced MMP-1 expression in dermal fibroblasts but through different pathways. As current strategies for reducing sun-related aging focused on filtering of light and use of antioxidants, future strategies design to reduce solar aging should also incorporate heat-induced aging into consideration. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  15. Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Secher, Jan Ole Bertelsen; Ceylan, Ahmet; Mazzoni, Gianluca

    2017-01-01

    Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to sh...

  16. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

    Science.gov (United States)

    Mizoshiri, N; Kishida, T; Yamamoto, K; Shirai, T; Terauchi, R; Tsuchida, S; Mori, Y; Ejima, A; Sato, Y; Arai, Y; Fujiwara, H; Yamamoto, T; Kanamura, N; Mazda, O; Kubo, T

    2015-11-27

    Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells

    Energy Technology Data Exchange (ETDEWEB)

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito, E-mail: y-ihara@wakayama-med.ac.jp

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells. - Highlights: • Hsc70 siRNA treatment suppressed the expression of Hsc70 but induced the expression of Hsp70 in NRK-49F cells. • Hsc70 siRNA treatment suppressed the activation of Smad2/3 in the cells treated with TGF-β. • Hsc70 interacted with Smad2/3 on stimulation with TGF-β in the cells. • Hsp70 did not influence the TGF-β-induced activation of Smad2/3 in the cells overexpressing Hsp70.

  18. Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

    Science.gov (United States)

    Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

    2016-01-01

    It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma. PMID:27468688

  19. Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of acid sphingomyelinase and inhibition of phospholipase A1

    International Nuclear Information System (INIS)

    Nassogne, Marie-Cecile; Lizarraga, Chantal; N'Kuli, Francisca; Van Bambeke, Francoise; Van Binst, Roger; Wallemacq, Pierre; Tulkens, Paul M.; Mingeot-Leclercq, Marie-Paule; Levade, Thierry; Courtoy, Pierre J.

    2004-01-01

    This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 μM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A 1 . At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A 1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in V max with identical K m . Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A 1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis

  20. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.

  1. Persistence of X-ray-induced chromosomal rearrangements in long-term cultures of human diploid fibroblasts

    International Nuclear Information System (INIS)

    Kano, Y.; Little, J.B.

    1984-01-01

    As part of a long-term study of mechanisms of human cell neoplastic transformation, the authors have examined the change in the frequencies of X-ray-induced chromosome rearrangements in density-inhibited human foreskin fibroblasts as a function of subculture time. In nonproliferating cells, the frequency of chromosomal aberrations declined within 24 to 48 hr but still remained at a relatively high level up to 43 days after irradiation. Aberrations disappeared rapidly, however, when the cells were allowed to proliferate, indicating that these lesions are lethal to dividing cells. The frequency of induced translocations, as determined by analysis of G-banded karyotypes, was dose dependent and remained stable up to 20 mean population doublings after irradiation. When subculture of density-inhibited cultures was delayed for 4 hr after irradiation (confluent holding), the frequency of chromosomal aberrations in the first mitosis declined, whereas the translocation frequencies at later passage were elevated as compared with cells subcultured immediately. This correlates with the reported increase in the frequency of transformation under similar conditions. These findings support the hypothesis that chromosomal rearrangements induced by DNA damage may be involved in the initiation of cancer

  2. Reversal of drug-induced gingival overgrowth by UV-mediated apoptosis of gingival fibroblasts - an in vitro study.

    Science.gov (United States)

    Ritchhart, Casey; Joy, Anita

    2018-05-01

    Gingival overgrowth (GO) is an undesirable result of certain drugs like Cyclosporine A (CsA). Histopathology of GO shows hyperplasia of gingival epithelium, expansion of connective tissue with increased collagen, or a combination. Factors such as age, gender, oral hygiene, duration, and dosage also influence onset and severity of GO. One of the mechanisms behind uncontrolled cell proliferation in drug-induced GO is inhibition of apoptotic pathways, with a consequent effect on normal cell turnover. Our objective was to determine if UV photo-treatment would activate apoptosis in the gingival fibroblast component. Human gingival fibroblast cells (HGF-1) were exposed to 200ng/ml or 400ng/ml CsA and maintained for 3, 6, and 9 days, followed by UV radiation for 2, 5, or 10min (N=6). Naïve (no CsA or UV), negative (UV, no CsA), and positive controls (CsA, no UV) were designated. Prior to UV treatment, growth media was replaced with 1M PBS to prevent absorption of UV radiation by serum proteins, and cells were incubated in growth media for 24h post-UV before processing for TUNEL assay, cell proliferation assays, or immunofluorescence. Data showed a temporal increase in proliferation of HGF-1 cells under the influence of CsA. The 200ng/ml dose was more effective in causing over-proliferation. UV treatment for 10min resulted in significant reduction in cell numbers, as evidenced by counts and proliferation assays. Our study is a first step to further evaluate UV-mediated apoptosis as a mechanism to control certain forms of GO. Copyright © 2018 Elsevier GmbH. All rights reserved.

  3. TGF-beta-induced early gene-1 overexpression promotes oxidative stress protection and actin cytoskeleton rearrangement in human skin fibroblasts.

    Science.gov (United States)

    Leduc, Chloe; Sobilo, Lauren; Toumi, Hechmi; Mondon, Philippe; Lespessailles, Eric; Ossant, Fédéric; Kurfurst, Robin; Pichon, Chantal

    2016-06-01

    Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Krüppel-like factor, was identified as a primary response gene for TGF-β. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (α-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK). TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65±5%) and exposure to ultraviolet further increased this effect (47±3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased α-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed. TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. This study enlightens the role of TIEG-1 role in skin biology. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

    Science.gov (United States)

    Tsukamoto, Akira; Higashiyama, Satoru; Yoshida, Kenji; Watanabe, Yoshiaki; Furukawa, Katsuko S; Ushida, Takashi

    2011-12-01

    An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Differential gene expression before and after ionizing radiation of subcutaneous fibroblasts identifies breast cancer patients resistant to radiation-induced fibrosis

    International Nuclear Information System (INIS)

    Alsner, Jan; Rodningen, Olaug K.; Overgaard, Jens

    2007-01-01

    Background and purpose: Differentially gene expression between patients with either very low or very high risk of radiation-induced fibrosis (RIF) in patient-derived fibroblasts after irradiation has previously been reported. In the present study, we are investigating the robustness of radiation-induced changes in gene expression in fibroblasts, whether differential expression is more pronounced when looking at the fold induction levels, taking into account the differences in background expression levels between patients, and whether there is a linear correlation between individual risk of RIF and changes in radiation-induced gene expression in fibroblasts. Material and methods: Gene expression was analysed by quantitative real-time PCR before and after a fractionated scheme with 3 x 3.5 Gy/3 days in fibroblasts derived from 26 patients with breast cancer treated with post-mastectomy radiotherapy. Results: Robust radiation-induced changes in gene expression were observed, with differential gene expression between low and high risk patients being most pronounced for the fold induction level ('after' value divided by 'before' value for each patient). When including patients with intermediate risk, there was no linear correlation between individual risk of RIF and differential expression of the genes investigated. Rather, differential gene expression could divide patients into two clearly separated groups, a larger, sensitive group and a smaller resistant group. Conclusions: Differential gene expression in irradiated fibroblasts might be an important tool in the identification of differences in the genetic background between patients with variable risk of RIF, and in the identification of new targets for prevention and intervention of the fibrotic process

  6. Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan, E-mail: shan_mou@126.com; Ni, Zhaohui, E-mail: doctor_nzh@126.com

    2015-09-04

    Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

  7. Plasma rich in growth factors (PRGF-Endoret) stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-beta1-Induced myodifferentiation.

    Science.gov (United States)

    Anitua, Eduardo; Sanchez, Mikel; Merayo-Lloves, Jesus; De la Fuente, Maria; Muruzabal, Francisco; Orive, Gorka

    2011-08-01

    Plasma rich in growth factors (PRGF-Endoret) technology is an autologous platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, and growth and differentiation. Because ocular surface wound healing is mediated by different growth factors, we decided to explore the potential of PRGF-Endoret technology in stimulating the biological processes related with fibroblast-induced tissue repair. Furthermore, the anti-fibrotic properties of this technology were also studied. Blood from healthy donors was collected, centrifuged and, whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were drawn off, avoiding the buffy coat. Primary human cells including keratocytes and conjunctival fibroblasts were used to perform the "in vitro" investigations. The potential of PRGF-Endoret in promoting wound healing was evaluated by means of a proliferation and migration assays. Fibroblast cells were induced to myofibroblast differentiation after the treatment with 2.5 ng/mL of TGF-β1. The capability of WP and F3 to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results show that this autologous approach significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, plasma rich in growth factors prevents and inhibits TGF-β1-induced myofibroblast differentiation. No differences were found between WP and F3 plasma fractions. These results suggest that PRGF-Endoret could reduce scarring while stimulating wound healing in ocular surface. F3 or whole plasma column show similar biological effects in keratocytes and conjunctival fibroblast cells.

  8. Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Rubio, Laura [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Spain); El Yamani, Naouale [Health Effects Laboratory-MILK, NILU-Norwegian Institute for Air Research, Kjeller (Norway); Kazimirova, Alena [Department of Biology, Slovak Medical University, Bratislava (Slovakia); Dusinska, Maria, E-mail: maria.dusinska@nilu.no [Health Effects Laboratory-MILK, NILU-Norwegian Institute for Air Research, Kjeller (Norway); Marcos, Ricard, E-mail: ricard.marcos@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Spain); CIBER Epidemiología y Salud Pública, Instituto de Salud Carlos III, Madrid (Spain)

    2016-04-15

    Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12 µg/cm{sup 2} At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure. - Highlights: • MWCNT were tested in V79 cells. • Cellular uptake of MWCNT was detected using TEM. • Intracellular ROS induction was observed after MWCNT exposure. • MWCNT induced a concentration-dependent increase of HPRT mutations.

  9. Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts

    International Nuclear Information System (INIS)

    Rubio, Laura; El Yamani, Naouale; Kazimirova, Alena; Dusinska, Maria; Marcos, Ricard

    2016-01-01

    Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12 µg/cm 2 At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure. - Highlights: • MWCNT were tested in V79 cells. • Cellular uptake of MWCNT was detected using TEM. • Intracellular ROS induction was observed after MWCNT exposure. • MWCNT induced a concentration-dependent increase of HPRT mutations.

  10. UVA-induced ROS generation inhibition by Oenothera paradoxa defatted seeds extract and subsequent cell death in human dermal fibroblasts.

    Science.gov (United States)

    Jaszewska, Edyta; Soin, Magdalena; Filipek, Agnieszka; Naruszewicz, Marek

    2013-09-05

    UVA radiation stimulates the production of reactive oxygen species (ROS), which react with lipids, proteins and other intracellular molecules leading to oxidative stress, cellular damage and ultimately cell death. There is, therefore, a growing need for substances exhibiting antioxidant activity, which may support repair mechanisms of the skin. This study evaluates the protective effect of the aqueous Oenothera paradoxa Hudziok defatted seeds extract, rich in polyphenolic compounds, against UVA (25 and 50J/cm(2))-induced changes in normal human dermal fibroblasts (NHDFs). The tested extract (0.1-10μg/ml) has decreased, in a concentration-dependent fashion, the UVA-induced release of lactate dehydrogenase (LDH) into the culture medium, the ROS production (with the use of 2',7'-dichlorodihydrofluorescein diacetate) and lipid peroxidation (utilizing redox reactions with ferrous ions) as compared to the control cells (incubated without the extract). Moreover, the extract increased the number of viable (calcein positive) cells decreasing the number of cells in late apoptosis (annexin V-FITC and propidium iodide positive). Thus our results show that O. paradoxa defatted seeds extract may be beneficial for the prevention of UVA skin damage. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1989-01-01

    We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage

  12. A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

    International Nuclear Information System (INIS)

    Pan Hui; Kantharia, Sarah; Jiang Hongliang; Chen Weiliam

    2011-01-01

    Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

  13. A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

    Energy Technology Data Exchange (ETDEWEB)

    Pan Hui; Kantharia, Sarah [Department of Biomedical Engineering, State University of New York-Stony Brook, Stony Brook, NY 11794-2580 (United States); Jiang Hongliang [Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027 (China); Chen Weiliam, E-mail: weiliam.chen@nyumc.org [Division of Wound Healing and Regenerative Medicine, Department of Surgery, New York University School of Medicine, New York, NY 10016 (United States)

    2011-12-15

    Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

  14. Fibroblast growth factor 10 protects neuron against oxygen–glucose deprivation injury through inducing heme oxygenase-1

    International Nuclear Information System (INIS)

    Li, Yong-Hua; Yang, Li-Ye; Chen, Wei; Li, Ying-Ke; Yuan, Hong-Bin

    2015-01-01

    Highlights: • FGF10 attenuates OGD induced injury in cortical neuron. • FGF10 reduces OGD triggered ROS level in cortical neuron. • FGF10 induces HO-1 expression upon OGD stimuli in cortical neuron. • Knockdown of HO-1 impairs the neuroprotection of FGF10 in OGD model. - Abstract: Fibroblast growth factors (FGFs) are a family of structurally related heparin-binding proteins with diverse biological functions. FGFs participate in mitogenesis, angiogenesis, cell proliferation, development, differentiation and cell migration. Here, we investigated the potential effect of FGF10, a member of FGFs, on neuron survival in oxygen–glucose deprivation (OGD) model. In primary cultured mouse cortical neurons upon OGD, FGF10 treatment (100 and 1000 ng/ml) attenuated the decrease of cell viability and rescued the LDH release. Tuj-1 immunocytochemistry assay showed that FGF10 promoted neuronal survival. Apoptosis assay with Annexin V + PI by flow cytometry demonstrated that FGF10 treatment reduced apoptotic cell proportion. Moreover, immunoblotting showed that FGF10 alleviated the cleaved caspase-3 upregulation caused by OGD. FGF10 treatment also depressed the OGD-induced increase of caspase-3, -8 and -9 activities. At last, we found FGF10 triggered heme oxygenase-1 (HO-1) protein expression rather than hypoxia-inducible factor-1 (HIF-1), AMP-activated protein kinase (AMPK) signaling and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling. Knockdown of HO-1 by siRNA partly abolished the neuroprotection of FGF10 in OGD model. In summary, our observations provide the first evidence for the neuroprotective function of FGF10 against ischemic neuronal injury and suggest that FGF10 may be a promising agent for treatment of ischemic stroke

  15. Hydrogen sulphide decreases IL-1β-induced activation of fibroblast-like synoviocytes from patients with osteoarthritis

    Science.gov (United States)

    Sieghart, Daniela; Liszt, Melissa; Wanivenhaus, Axel; Bröll, Hans; Kiener, Hans; Klösch, Burkhard; Steiner, Günter

    2015-01-01

    Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1β and treated with the H2S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1β-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1β-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2S partially antagonizes IL-1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA. PMID:25312962

  16. Fibroblast growth factor 10 protects neuron against oxygen–glucose deprivation injury through inducing heme oxygenase-1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong-Hua; Yang, Li-Ye; Chen, Wei; Li, Ying-Ke, E-mail: liyingke6f@126.com; Yuan, Hong-Bin, E-mail: yuanhongbin6f@126.com

    2015-01-02

    Highlights: • FGF10 attenuates OGD induced injury in cortical neuron. • FGF10 reduces OGD triggered ROS level in cortical neuron. • FGF10 induces HO-1 expression upon OGD stimuli in cortical neuron. • Knockdown of HO-1 impairs the neuroprotection of FGF10 in OGD model. - Abstract: Fibroblast growth factors (FGFs) are a family of structurally related heparin-binding proteins with diverse biological functions. FGFs participate in mitogenesis, angiogenesis, cell proliferation, development, differentiation and cell migration. Here, we investigated the potential effect of FGF10, a member of FGFs, on neuron survival in oxygen–glucose deprivation (OGD) model. In primary cultured mouse cortical neurons upon OGD, FGF10 treatment (100 and 1000 ng/ml) attenuated the decrease of cell viability and rescued the LDH release. Tuj-1 immunocytochemistry assay showed that FGF10 promoted neuronal survival. Apoptosis assay with Annexin V + PI by flow cytometry demonstrated that FGF10 treatment reduced apoptotic cell proportion. Moreover, immunoblotting showed that FGF10 alleviated the cleaved caspase-3 upregulation caused by OGD. FGF10 treatment also depressed the OGD-induced increase of caspase-3, -8 and -9 activities. At last, we found FGF10 triggered heme oxygenase-1 (HO-1) protein expression rather than hypoxia-inducible factor-1 (HIF-1), AMP-activated protein kinase (AMPK) signaling and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling. Knockdown of HO-1 by siRNA partly abolished the neuroprotection of FGF10 in OGD model. In summary, our observations provide the first evidence for the neuroprotective function of FGF10 against ischemic neuronal injury and suggest that FGF10 may be a promising agent for treatment of ischemic stroke.

  17. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    Energy Technology Data Exchange (ETDEWEB)

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin, E-mail: salah.ahmed@wsu.edu

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

  18. Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

    Science.gov (United States)

    Chen, Jung-Tsu; Wang, Chen-Ying; Chen, Min-Huey

    2018-01-13

    Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. Copyright © 2018. Published by Elsevier B.V.

  19. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yuliati

    2015-12-01

    Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  20. [Malignant transformation of human fibroblasts by neutrons and by gamma radiation: Relationship to mutations induced

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1993-12-31

    A brief overview if provided of selected reports presented at the International Symposium on Molecular Mechanisms of Radiation- and Chemical Carcinogen-Induced Cell Transformation held at Mackinac Island, Michigan on September 19-23, 1993.

  1. Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured normal human dermal fibroblasts

    DEFF Research Database (Denmark)

    Almqvist, Sofia; Werthén, Maria; Johansson, Anna

    2010-01-01

    Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100...

  2. Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

    Science.gov (United States)

    Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

    2014-05-02

    The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

  3. Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients.

    Science.gov (United States)

    Maggi, Laura; Margheri, Francesca; Luciani, Cristina; Capone, Manuela; Rossi, Maria Caterina; Chillà, Anastasia; Santarlasci, Veronica; Mazzoni, Alessio; Cimaz, Rolando; Liotta, Francesco; Maggi, Enrico; Cosmi, Lorenzo; Del Rosso, Mario; Annunziato, Francesco

    2016-01-01

    This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

  4. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  5. Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis.

    Science.gov (United States)

    Lin, Jinpiao; Zhou, Zhou; Huo, Rongfen; Xiao, Lianbo; Ouyang, Guilin; Wang, Li; Sun, Yue; Shen, Baihua; Li, Dangsheng; Li, Ningli

    2012-06-01

    Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

  6. MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion.

    Science.gov (United States)

    Park, Woo Hyun; Kim, Suhn Hee

    2012-04-01

    MG132 as a proteasome inhibitor can induce apoptotic cell death in lung cancer cells. However, little is known about the toxicological cellular effects of MG132 on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). MG132 induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). MG132 increased ROS levels and GSH-depleted cell numbers in HPF cells. Both antioxidants, NAC and vitamin C, prevented growth inhibition, death and MMP (∆ψm) loss in MG132-treated HPF cells and also attenuated ROS levels in these cells. BSO showed a strong increase in ROS levels in MG132-treated HPF cells and slightly enhanced the growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion. In addition, NAC decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. Furthermore, superoxide dismutase (SOD) 2, catalase (CTX) and GSH peroxidase (GPX) siRNAs enhanced HPF cell death by MG132, which was not correlated with ROS and GSH level changes. In conclusion, MG132 induced the growth inhibition and death of HPF cells, which were accompanied by increasing ROS levels and GSH depletion. Both NAC and vitamin C attenuated HPF cell death by MG132, whereas BSO slightly enhanced the death.

  7. Radio-induced superficial fibrosis: investigation of the activation mechanisms of the myo-fibroblast and characterization of the cicatricial epidermis

    International Nuclear Information System (INIS)

    Sivan, Virginie

    2001-01-01

    Whereas radio-induced cutaneous fibrosis is one of the frequent after-effects of accidental and therapeutic irradiations, this research thesis addresses the mechanisms which govern the activation of the myo-fibroblast. After some results obtained on cells from a radio-induced fibrosis on swine cells, the author proposes a signalling alteration as a mechanism. In a model a reconstructed skin, the author shows that myo-fibroblasts are a direct target of Superoxide Dismutase (SOD), and respond to this anti-fibrosis agent by a phenotype reversion. She reports the molecular characterization of epidermis of fibro-necrosis human lesions induced by an accidental or therapeutic irradiation. This leads to a better understanding of the role of the myo-fibroblast during the development and regression of fibrosis. Besides, the author shows that an alteration of the epidermis adjacent to dermis is developing in parallel with the fibrosis process. This suggests an active contribution of keratinocytes during the development of this radio-induced after-effect [fr

  8. Chronic Low Dose Rate Ionizing Radiation Exposure Induces Premature Senescence in Human Fibroblasts that Correlates with Up Regulation of Proteins Involved in Protection against Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Olga Loseva

    2014-07-01

    Full Text Available The risks of non-cancerous diseases associated with exposure to low doses of radiation are at present not validated by epidemiological data, and pose a great challenge to the scientific community of radiation protection research. Here, we show that premature senescence is induced in human fibroblasts when exposed to chronic low dose rate (LDR exposure (5 or 15 mGy/h of gamma rays from a 137Cs source. Using a proteomic approach we determined differentially expressed proteins in cells after chronic LDR radiation compared to control cells. We identified numerous proteins involved in protection against oxidative stress, suggesting that these pathways protect against premature senescence. In order to further study the role of oxidative stress for radiation induced premature senescence, we also used human fibroblasts, isolated from a patient with a congenital deficiency in glutathione synthetase (GS. We found that these GS deficient cells entered premature senescence after a significantly shorter time of chronic LDR exposure as compared to the GS proficient cells. In conclusion, we show that chronic LDR exposure induces premature senescence in human fibroblasts, and propose that a stress induced increase in reactive oxygen species (ROS is mechanistically involved.

  9. Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ziqing Liu

    2016-03-01

    Full Text Available Direct conversion of fibroblasts into induced cardiomyocytes (iCMs offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome

  10. Effects of budesonide and formoterol on eosinophil activation induced by human lung fibroblasts

    NARCIS (Netherlands)

    Spoelstra, FM; Kauffman, HF; Hovenga, H; Noordhoek, JA; de Monchy, JGR; Postma, DS

    2000-01-01

    Budesonide and formoterol are extensively used in current asthma therapy. Budesonide is known as potent antiinflammatory agent and formoterol also appears to have some antiinflammatory properties. We investigated inhibitory effects of these drugs on eosinophil activation in vitro as induced by

  11. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  12. Tumor-Induced Osteomalacia Caused by Primary Fibroblast Growth Factor 23 Secreting Neoplasm in Axial Skeleton: A Case Report

    Directory of Open Access Journals (Sweden)

    Gunjan Y. Gandhi

    2012-01-01

    Full Text Available We report the case of a 66-year-old woman with tumor-induced osteomalacia (TIO caused by fibroblast growth factor 23 (FGF-23 secreting mesenchymal tumor localized in a lumbar vertebra and review other cases localized to the axial skeleton. She presented with nontraumatic low back pain and spontaneous bilateral femur fractures. Laboratory testing was remarkable for low serum phosphorus, phosphaturia, and significantly elevated serum FGF-23 level. Magnetic resonance imaging (MRI of the lumbar spine showed a focal lesion in the L-4 vertebra which was hypermetabolic on positron emission tomography (PET scan. A computed tomography (CT guided needle biopsy showed a low grade spindle cell neoplasm with positive FGF-23 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR, confirming the diagnosis of a phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT. The patient elected to have surgery involving anterior resection of L-4 vertebra with subsequent normalization of serum phosphorus. Including the present case, we identified 12 cases of neoplasms localized to spine causing TIO. To our knowledge, this paper represents the first documented case of lumbar vertebra PMT causing TIO. TIO is a rare metabolic bone disorder that carries a favorable prognosis. When a lesion is identifiable, surgical intervention is typically curative.

  13. Distribution of u.v.-induced repair events in higher-order chromatin loops in human and hamster fibroblasts

    International Nuclear Information System (INIS)

    Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.; Kesteren, A.C. van; Bussmann, C.J.M.

    1986-01-01

    The repair of u.v.-induced damage in human and rodent cells was investigated at the level of DNA loops attached to the nuclear matrix. After 2 h post-u.v. incubation, DNase I digestion studies revealed a 3- to 4-fold enrichment of repair-labeled DNA at the nuclear matrix in four xeroderma pigmentosum cell strains belonging to complementation group C. Two xeroderma pigmentosum cell strains of complementation group D and Syrian hamster embryonic cells, as well as in HeLa cells and normal human fibroblasts, no enrichment of repair-labeled DNA at the nuclear matrix was observed. Visualization of repair events in DNA loops by autoradiography of DNA halo - matrix structures confirmed the biochemical observations. The presence or absence of preferential repair of nuclear matrix-associated DNA paralleled the presence or absence of inhomogeneity in the distribution of T4 endonuclease-V-sensitive sites. In xeroderma pigmentosum cells of complementation group C showed that after 2 h post-u.v. incubation, repair events were found at both attachment sites in a limited number of loops and that large domains of loops were not subjected to repair. (author)

  14. GSK126 (EZH2 inhibitor) interferes with ultraviolet A radiation-induced photoaging of human skin fibroblast cells

    Science.gov (United States)

    Qin, Haiyan; Zhang, Guang; Zhang, Lianbo

    2018-01-01

    Polycomb group genes (PcG) encode chromatin modification proteins that are involved in the epigenetic regulation of cell differentiation, proliferation and the aging processes. The key subunit of the PcG complex, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), has a central role in a variety of mechanisms, such as the formation of chromatin structure, gene expression regulation and DNA damage. In the present study, ultraviolet A (UVA) was used to radiate human dermal fibroblasts in order to construct a photo-aged cell model. Subsequently, the cell viability assay, Hoechst staining, apoptosis detection using flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining and erythrocyte exclusion experiments were performed. GSK126, a histone methylation enzyme inhibitor of EZH2, was used as an experimental factor. Results suggested that GSK126 downregulated the mRNA expression levels of EZH2 and upregulated the mRNA expression levels of BMI-1. Notably, GSK126 affected the transcription of various photoaging-related genes and thus protected against photoaging induced by UVA radiation. PMID:29545866

  15. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  16. Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Jasper Foolen

    Full Text Available Generating and maintaining gradients of cell density and extracellular matrix (ECM components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. 'compact and adsorbed to collagen' versus 'extended and fibrillar' fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin's contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs and floxed equivalents (Fnf/f MEFs, in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments. In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen

  17. In vitro growth potential of fibroblasts isolated from pigs with radiation-induced fibrosis

    International Nuclear Information System (INIS)

    Martin, M.; Remy, J.; Daburon, F.

    1986-01-01

    Degenerative processes were studied in pig muscles irradiated with single doses of 30 or 40 Gy. Damaged muscle was gradually replaced by an invasive fibrotic tissue. As a control, surgical muscle exeresis was performed of the same size as the radiation-induced lesions at the same anatomical site. Primary cultures were set up comprising cells freshly extracted from normal dermis, or from tissue exhibiting either normal wound fibrosis or radiation-induced fibrosis. The growth potential of cells taken from the latter region far exceeded that of the two other types; attachment efficiency was higher, and fibronectin was detected early by immunofluorescence. These in vivo and in vitro observations imply that a pathological repair process occurs after localized irradiation. (author)

  18. Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

    Science.gov (United States)

    Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

    2010-04-09

    Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

  19. Protective effect of basic fibroblast growth factor on retinal injury induced by argon laser photocoagulation

    International Nuclear Information System (INIS)

    Chen, P; San, Q; Wang, C Z; Yang, Z F; Kang, H X; Qian, H W; Zhang, C P

    2010-01-01

    Laser photocoagulation treatment is often complicated by a side effect of visual impairment, which is caused by the unavoidable laser-induced retinal destruction. At present no specific is found to cure this retinopathy. The aim of this study was to observe the neuroprotective effect of bFGF on laser-induced retinal injury. Chinchilla rabbits were divided into three groups and argon laser lesions were created in the retinas. Then bFGF or dexamethasone, a widely used ophthalmic preparation, or saline was given severally by retrobulbar injection. The retinal lesions were evaluated histologically and morphometrically, and visual function was examined by ERG. The results showed that bFGF administration better preserved morphology of retinal photoreceptors and significantly diminished the area of the lesions. Furthermore, bFGF promoted the restoration of the ERG b-wave amplitude. In rabbits treated with dexamethasone, however, the lesions showed almost no ameliorative changes. This is the first study to investigate the potential role of bFGF as a remedial agent in laser photocoagulation treatment. These findings suggest that bFGF has significant neuroprotective properties in the retina and this type of neuroprotection may be of clinical significance in reducing iatrogenic laser-induced retinal injuries in humans

  20. Peroxiredoxin 5 Protects TGF-β Induced Fibrosis by Inhibiting Stat3 Activation in Rat Kidney Interstitial Fibroblast Cells.

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    Hoon-In Choi

    Full Text Available Renal fibrosis is a common final pathway of end-stage kidney disease which is induced by aberrant accumulation of myofibroblasts. This process is triggered by reactive oxygen species (ROS and proinflammatory cytokines generated by various source of injured kidney cells. Peroxiredoxin 5 (Prdx5 is a thiol-dependent peroxidase that reduces oxidative stress by catalyzing intramolecular disulfide bonds. Along with its antioxidant effects, expression level of Prdx5 also was involved in inflammatory regulation by immune stimuli. However, the physiological effects and the underlying mechanisms of Prdx5 in renal fibrosis have not been fully characterized. Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO for 1 or 7 days. For the in vitro model, NRK49F cells, a rat kidney interstitial fibroblast cell lines, were treated with transforming growth factor β (TGF-β for 0, 1, 3, or 5 days. To access the involvement of its peroxidase activity in TGF-β induced renal fibrosis, wild type Prdx5 (WT and double mutant Prdx5 (DM, converted two active site cysteines at Cys 48 and Cys 152 residue to serine, were transiently expressed in NRK49F cells. The protein expression of Prdx5 was reduced in UUO kidneys. Upregulation of fibrotic markers, such as fibronectin and alpha-smooth muscle actin (α-SMA, declined at 5 days in time point of higher Prdx5 expression in TGF-β treated NRK49F cells. The overexpression of wild type Prdx5 by transient transfection in NRK49F cells attenuated the TGF-β induced upregulation of fibronectin and α-SMA. On the other hand, the transient transfection of double mutant Prdx5 did not prevent the activation of fibrotic markers. Overexpression of Prdx5 also suppressed the TGF-β induced upregulation of Stat3 phosphorylation, while phosphorylation of Smad 2/3 was unchanged. In conclusion, Prdx5 protects TGF-β induced fibrosis in NRK49F cells by modulating Stat3 activation in a peroxidase activity dependent manner.

  1. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

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    Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

    2016-05-06

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  2. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Yin, Xiaohui; Li, Yang; Li, Jingwen; Li, Peng; Liu, Yinan; Wen, Jinhua; Luan, Qingxian

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  3. Tamaractam, a New Bioactive Lactam from Tamarix ramosissima, Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

    Science.gov (United States)

    Yao, Yao; Jiang, Cheng-Shuai; Sun, Na; Li, Wei-Qi; Niu, Yang; Han, Huai-Qin; Miao, Zhen-Hua; Zhao, Xun-Xia; Zhao, Jing; Li, Juan

    2017-01-10

    Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA) treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam ( 1 ), together with the previously reported compounds cis - N -feruloyl-3- O -methyldopamine ( 2 ) and trans - N -feruloyl-3- O -methyldopamine ( 3 ). The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS) and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS) were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2- H -tetrazolium bromide (MTT) assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G₁ fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G₁ fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima .

  4. Tamaractam, a New Bioactive Lactam from Tamarix ramosissima, Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

    Directory of Open Access Journals (Sweden)

    Yao Yao

    2017-01-01

    Full Text Available Chemical investigation of Tamarix ramosissima Ledeb, a traditional herbal medicine used for rheumatoid arthritis (RA treatment in northwest China, led to the discovery of a new phenolic aromatic rings substituted lactam, tamaractam (1, together with the previously reported compounds cis-N-feruloyl-3-O-methyldopamine (2 and trans-N-feruloyl-3-O-methyldopamine (3. The structures of the compounds were determined by high resolution electrospray ionization mass spectroscopy (HRESIMS and 1D and 2D-NMR experiments, as well as comparison with the literature data. The effects of the three compounds on the viability of RA fibroblast-like synoviocytes (RA-FLS were assessed by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay. Pro-apoptosis effect of compound 1 in RA-FLS was further investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, activated caspase-3/7 level assessment using luminescence assay, and sub-G1 fraction measurement using flow cytometry. It was found that these three compounds displayed variable proliferation inhibitory activity in RA-FLS, and compound 1 exhibited the strongest effect. Compound 1 could remarkably induce cellular apoptosis of RA-FLS, increase activated caspase-3/7 levels, and significantly increase sub-G1 fraction in the cell cycle. The results suggested that compound 1 may inhibit the proliferation of RA-FLS through apoptosis-inducing effect, and these compounds may contribute to the anti-RA effect of T. ramosissima.

  5. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

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    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  6. Human amnion-derived mesenchymal stem cells protect against UVA irradiation-induced human dermal fibroblast senescence, in vitro

    Science.gov (United States)

    Zhang, Chunli; Yuchi, Haishen; Sun, Lu; Zhou, Xiaoli; Lin, Jinde

    2017-01-01

    The aim of the present study was to determine if human amnion-derived mesenchymal stem cells (HAMSCs) exert a protective effect on ultraviolet A (UVA) irradiation-induced human dermal fibroblast (HDF) senescence. A senescence model was constructed as follows: HDFs (104–106 cells/well) were cultured in a six-well plate in vitro and then exposed to UVA irradiation at 9 J/cm2 for 30 min. Following the irradiation period, HDFs were co-cultured with HAMSCs, which were seeded on transwells. A total of 72 h following the co-culturing, senescence-associated β-galactosidase staining was performed and reactive oxygen species (ROS) content and mitochondrial membrane potential (Δψm) were detected in the HDFs via flow cytometric analysis. The results demonstrated that the percentage of HDFs, detected via staining with X-gal, were markedly decreased when co-cultured with human HAMSCs, compared with the group that were not co-cultured. The ROS content was decreased and the mitochondrial membrane potential (Δψm) recovered in cells treated with UVA and HAMSCs, compared with that of cells treated with UVA alone. Reverse transcription-quantitative polymerase chain reaction revealed the significant effects of HAMSCs on the HDF senescence marker genes p53 and matrix metalloproteinase-1 mRNA expression. In addition to this, western blot analysis verified the effects of HAMSCs on UVA induced senescence, providing a foundation for novel regenerative therapeutic methods. Furthermore, the results suggested that activation of the extracellular-signal regulated kinase 1/2 mitogen activated protein kinase signal transduction pathway, is essential for the HAMSC-mediated UVA protective effects. The decrease in ROS content additionally indicated that HAMSCs may exhibit the potential to treat oxidative stress-mediated UVA skin senescence in the future. PMID:28627622

  7. Poly(I:C) induces expressions of MMP-1, -2, and -3 through various signaling pathways including IRF3 in human skin fibroblasts.

    Science.gov (United States)

    Yao, Cheng; Lee, Dong Hun; Oh, Jang-Hee; Kim, Min-Kyoung; Kim, Kyu Han; Park, Chi-Hyun; Chung, Jin Ho

    2015-10-01

    Ultraviolet (UV) irradiation can result in premature skin aging (photoaging) which is characterized by decreased expression of collagen and increased expression of matrix metalloproteinases (MMPs). Double-stranded RNAs (dsRNAs) can be generated at various conditions including virally infected cells or UV-damaged skin cells. Recent studies have shown that a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), can reduce procollagen expression in human skin fibroblasts. However, little is known about the effect of poly(I:C) on the expression of MMPs in skin fibroblasts and its underlying mechanisms. We examined the effect of poly(I:C) on MMP-1, -2, and -3 expressions in human skin fibroblasts. Then, we further explored the underlying signaling pathways involved in the processes. Human skin fibroblasts were treated with poly(I:C) for the indicated times in the presence or the absence of various chemical inhibitors or small interfering RNAs (siRNAs) at the indicated concentrations. Protein and mRNA levels of various target molecules were examined by Western blotting and quantitative real-time PCR, respectively. Poly(I:C) induced MMP-1, -2, and -3 expressions, which were dependent on TLR3. Poly(I:C) also induced activations of the mitogen-activated protein kinases (MAPKs), the nuclear factor-kappaB (NF-κB) and the interferon regulatory factor 3 (IRF3) pathways. By using specific inhibitors, we found that poly(I:C)-induced expressions of MMP-1, -2, and -3 were differentially regulated by these signaling pathways. In particular, we found that the inhibition of IRF3 signaling pathways attenuated poly(I:C)-induced expressions of all the three MMPs. Our data show that the expressions of MMP-1, -2, and -3 are induced by poly(I:C) through various signaling pathways in human skin fibroblasts and suggest that TLR3 and/or IRF3 may be good targets for regulating the expressions of MMP-1, -2, and -3 induced by dsRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights

  8. Protective role of Nrf2 against mechanical-stretch-induced apoptosis in mouse fibroblasts: a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence.

    Science.gov (United States)

    Li, Qiannan; Li, Bingshu; Liu, Cheng; Wang, Linlin; Tang, Jianming; Hong, Li

    2018-01-10

    We investigated the protective effect and underlying molecular mechanism of nuclear factor-E2-related factor 2 (Nrf2) against mechanical-stretch-induced apoptosis in mouse fibroblasts. Normal cells, Nrf2 silencing cells, and Nrf2 overexpressing cells were respectively divided into two groups-nonintervention and cyclic mechanical strain (CMS)-subjected to CMS of 5333 μ (1.0 Hz for 4 h), six groups in total (control, CMS, shNfe212, shNfe212 + CMS, LV-shNfe212, and LV-shNfe212 + CMS). After treatment, cell apoptosis; cell-cycle distribution; expressions of Nrf2, Bax, Bcl-2, Cyt-C, caspase-3, caspase-9, cleaved-caspase-3, and cleaved-caspase-9; mitochondrial membrane potential (ΔΨm); reactive oxygen species (ROS); and malondialdehyde (MDA) levels were measured. Thirty virgin female C57BL/6 mice were divided into two groups: control (without intervention) and vaginal distension (VD) groups, which underwent VD for 1 h with an 8-mm dilator (0.3 ml saline). Leak-point pressure (LPP) was tested on day 7 after VD; Nrf2 expression, apoptosis, and MDA levels were then measured in urethra and anterior vaginal wall. Mechanical stretch decreased Nrf2 messenger RNA (mRNA) and protein expressions. Overexpression of Nrf2 alleviated mechanical-stretch-induced cell apoptosis; S-phase arrest of cell cycle; up-regulation of Bax, cytochrome C (Cyt-C), ROS, MDA, ratio of cleaved-caspase-3/caspase-3 and cleaved-caspase-9/caspase-9; and exacerbated the decrease of Bcl2 and ΔΨm in L929 cells. On the contrary, silencing of Nrf2 showed opposite effects. Besides, VD reduced LPP levels and Nrf2 expression and increased cell apoptosis and MDA generation in the urethra and anterior vaginal wall. Nrf2 exhibits a protective role against mechanical-stretch -induced apoptosis on mouse fibroblasts, which might indicate a potential therapeutic target of mechanical-trauma-induced stress urinary incontinence (SUI).

  9. Fibroblastic rheumatism

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    Jyoti Ranjan Parida

    2017-01-01

    Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

  10. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

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    Xia Luo

    2015-01-01

    Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

  11. FGF-2 expression and the amount of fibroblast in the incised wounds of Rattus norvegicus rats induced with Mauli banana (Musa acuminata stem extract

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    Didit Aspriyanto

    2017-09-01

    Full Text Available Background: Traditional wound treatment using herbal medicine is thought to maintain the health of families and society in general economically, effectively, and efficiently without inducing side effects. One genus of plant that can be used as a traditional medicine is the Mauli banana, indigenous to South Borneo. Mauli banana stem contains bioactive compounds, most of which are tannins along with ascorbic acid, saponin, β-carotene, flavonoids, lycopene, alkaloids, and flavonoids. Tanin has antibacterial and antioxidant effects at low concentrations, as wells as antifungal ones at high concentrations. Purpose: This study aimed to analyze the effects of Mauli banana stem extract at concentrations of 25%, 37.5%, and 50% on the quality of incised wound healing in male Rattus norvegicus rats by assessing FGF-2 expression and fibroblast concentration on days 3 and 7. Methods: This research represented an experimental laboratory-based investigation involving 32 rats of the Rattus norvegicus strain aged 2-2.5 months old. Sampling was performed using a simple random sampling technique since the research population was considered homogeneous and divided into 8 treatment groups (C3, M3-25, M3-37.5, M3-50, C7, M7-25, M7-37.5, M7-50. The rats in each group were anesthetized before their back was incised with length and width of 15x15mm with a depth of 2mm. Gel hydroxy propyl cellulose medium (HPMC was applied to the incised wound of each rat in the control group, while stem Mauli banana extract was applied to that of each rat in the treatment groups three times a day at an interval of 6-8 hours. On day 3, four rats from each group were sacrificed, while, in the remaining groups, the same procedure was performed until day 7, at which point they (8 groups were sacrificed for HE examination in order to assess the amount of fibroblast and for IHC examination to examine FGF-2 expression. Data regarding FGF-2 expression and the amount of fibroblast were analysed

  12. The Janus kinase inhibitor tofacitinib inhibits TNF-α-induced gliostatin expression in rheumatoid fibroblast-like synoviocytes.

    Science.gov (United States)

    Kawaguchi, Yohei; Waguri-Nagaya, Yuko; Tatematsu, Naoe; Oguri, Yusuke; Kobayashi, Masaaki; Nozaki, Masahiro; Asai, Kiyofumi; Aoyama, Mineyoshi; Otsuka, Takanobu

    2018-01-15

    Gliostatin (GLS) is known to have angiogenic and arthritogenic activity, and GLS expression levels in serum from patients with rheumatoid arthritis (RA) are significantly correlated with the disease activity. Tofacitinib is a novel oral Janus kinase (JAK) inhibitor and is effective in treating RA. However, the mechanism of action of tofacitinib in fibroblast-like synoviocytes (FLSs) has not been elucidated. The purpose of this study was to investigate the modulatory effects of tofacitinib on serum GLS levels in patients with RA and GLS production in FLSs derived from patients with RA. Six patients with RA who had failed therapy with at least one TNF inhibitor and were receiving tofacitinib therapy were included in the study. Serum samples were collected to measure CRP, MMP-3 and GLS expression. FLSs derived from patients with RA were cultured and stimulated by TNFα with or without tofacitinib. GLS expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR), EIA and immunocytochemistry, and signal transducer and activator of transcription (STAT) protein phosphorylation levels were determined by western blotting. Treatment with tofacitinib decreased serum GLS levels in all patients. GLS mRNA and protein expression levels were significantly increased by treatment with TNF-α alone, and these increases were suppressed by treatment with tofacitinib, which also inhibited TNF-α-induced STAT1 phosphorylation. JAK/STAT activation plays a pivotal role in TNF-α-mediated GLS up-regulation in RA. Suppression of GLS expression in FLSs has been suggested to be one of the mechanisms through which tofacitinib exerts its anti-inflammatory effects.

  13. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jing Lu

    Full Text Available Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

  14. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts.

    Science.gov (United States)

    Chen, Joseph C; Johnson, Brittni A; Erikson, David W; Piltonen, Terhi T; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C; Greene, Warner C; Giudice, Linda C; Roan, Nadia R

    2014-06-01

    How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11

  15. Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.

    Science.gov (United States)

    Li, Yu; Wong, Kimberly; Giles, Amber; Jiang, Jianwei; Lee, Jong Woo; Adams, Andrew C; Kharitonenkov, Alexei; Yang, Qin; Gao, Bin; Guarente, Leonard; Zang, Mengwei

    2014-02-01

    The hepatocyte-derived hormone fibroblast growth factor 21 (FGF21) is a hormone-like regulator of metabolism. The nicotinamide adenine dinucleotide-dependent deacetylase SIRT1 regulates fatty acid metabolism through multiple nutrient sensors. Hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese mice. We investigated mechanisms by which SIRT1 controls hepatic steatosis in mice. Liver-specific SIRT1 knockout (SIRT1 LKO) mice and their wild-type littermates (controls) were divided into groups that were placed on a normal chow diet, fasted for 24 hours, or fasted for 24 hours and then fed for 6 hours. Liver tissues were collected and analyzed by histologic examination, gene expression profiling, and real-time polymerase chain reaction assays. Human HepG2 cells were incubated with pharmacologic activators of SIRT1 (resveratrol or SRT1720) and mitochondrion oxidation consumption rate and immunoblot analyses were performed. FGF21 was overexpressed in SIRT1 LKO mice using an adenoviral vector. Energy expenditure was assessed by indirect calorimetry. Prolonged fasting induced lipid deposition in livers of control mice, but severe hepatic steatosis in SIRT1 LKO mice. Gene expression analysis showed that fasting up-regulated FGF21 in livers of control mice but not in SIRT1 LKO mice. Decreased hepatic and circulating levels of FGF21 in fasted SIRT1 LKO mice were associated with reduced hepatic expression of genes involved in fatty acid oxidation and ketogenesis, and increased expression of genes that control lipogenesis, compared with fasted control mice. Resveratrol or SRT1720 each increased the transcriptional activity of the FGF21 promoter (-2070/+117) and levels of FGF21 messenger RNA and protein in HepG2 cells. Surprisingly, SIRT1 LKO mice developed late-onset obesity with impaired whole-body energy expenditure. Hepatic overexpression of FGF21 in SIRT1 LKO mice increased the expression of genes that regulate fatty acid oxidation, decreased

  16. Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, Fumiaki, E-mail: f_naka@nirs.go.jp [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Umeda, Sachiko [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Yasuda, Takeshi [Department of Radiation Emergency Medicine, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba (Japan); Asada, Masahiro; Motomura, Kaori; Suzuki, Masashi [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Zakrzewska, Malgorzata [Faculty of Biotechnology, University of Wroclaw (Poland); Imamura, Toru [Signaling Molecules Research Laboratory, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki (Japan); Imai, Takashi [Advanced Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan)

    2013-02-01

    Purpose: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. Methods and Materials: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with {gamma}-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. Results: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. Conclusion: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal

  17. Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xinyue Liang

    2016-01-01

    Full Text Available Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR. In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK/extracellular signal-regulated kinase (ERK and phosphatidylinositol 3′ -kinase(PI3K-Akt (PI3K/AKT phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy. In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

  18. Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

    DEFF Research Database (Denmark)

    Maynard, Scott P; Miller, Richard A

    2006-01-01

    Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwar...... in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice.......Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf...

  19. Curcumin attenuates inflammatory response in IL-1beta-induced human synovial fibroblasts and collagen-induced arthritis in mouse model.

    Science.gov (United States)

    Moon, Dong-Oh; Kim, Mun-Ok; Choi, Yung Hyun; Park, Yung-Min; Kim, Gi-Young

    2010-05-01

    Curcumin, a major component of turmeric, has been shown to exhibit anti-oxidant and anti-inflammatory activities. The present study was performed to determine whether curcumin is efficacious against both collagen-induced arthritis (CIA) in mice and IL-1beta-induced activation in fibroblast-like synoviocytes (FLSs). DBA/1 mice were immunized with bovine type II collagen (CII) and treated with curcumin every other day for 2weeks after the initial immunization. For arthritis, we evaluated the incidence of disease and used an arthritis index based on paw thickness. In vitro proliferation of CII- or concanavalin A-induced splenic T cells was examined using IFN-gamma production. Pro-inflammatory cytokines TNF-alpha and IL-1beta were examined in the mouse ankle joint and serum IgG1 and IgG2a isotypes were analyzed. The expression levels of prostaglandin E(2) (PGE(2)), cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) in human FLSs were also determined. The results showed that compared with untreated CIA mice, curcumin-treated mice downregulated clinical arthritis score, the proliferation of splenic T cells, expression levels of TNF-alpha and IL-1beta in the ankle joint, and expression levels of IgG2a in serum. Additionally, by altering nuclear factor (NF)-kappaB transcription activity in FLSs, curcumin inhibited PGE(2) production, COX-2 expression, and MMP secretion. These results suggest that curcumin can effectively suppress inflammatory response by inhibiting pro-inflammatory mediators and regulating humoral and cellular immune responses. Copyright 2010 Elsevier B.V. All rights reserved.

  20. A correlation between residual radiation-induced DNA double-strand breaks in cultured fibroblasts and late radiotherapy reactions in breast cancer patients

    International Nuclear Information System (INIS)

    Kiltie, A.E.; Ryan, A.J.; Swindell, R.; Barber, J.B.P.; West, C.M.L.; Magee, B.; Hendry, J.H.

    1999-01-01

    Background and purpose: Prediction of late normal tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Measurement of residual DNA double strand breaks using pulsed field gel electrophoresis (PFGE) shows promise in this field. The aim of this study was to test the predictive potential of PFGE in a group of retrospectively studied breast cancer patients.Materials and methods: Thirty nine patients, treated uniformly for breast cancer 9-15 years previously, with excision of the tumour and radiotherapy to the breast and drainage areas, were assessed clinically using the LENT SOMA scale, and a 5-mm punch biopsy taken from the buttock. Fibroblast cell strains were established and used to study residual DNA double strand breaks, using PFGE.Results: There were significant correlations between the DNA assay results and the fibrosis score (r s =0.46; P=0.003), the combined fibrosis and retraction score (r s =0.45, P=0.004) and the overall LENT score (r s =0.43; P=0.006). Using polychotomous logistic regression, the fibroblast DNA assay result was an independent prognostic factor for fibrosis severity.Conclusions: There is a relationship between residual radiation-induced DNA damage in fibroblasts and the severity of the late normal tissue damage seen in the patients from whom the cells were cultured. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  1. Activity of ethanolic extracts leaves of Machaerium floribundum against acne-inducing bacteria, and their cytoprotective and antioxidant effects on fibroblast

    Directory of Open Access Journals (Sweden)

    Lorena Díaz

    2011-08-01

    Full Text Available Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus have been recognized as the bacteria that are involved in the inflammatory process of acne, while oxidants and antioxidants are involved in the repair of cutaneous tissue affected. In this study an evaluation was made of the antibacterial effect by the agar diffusion and broth dilution method, the cytoprotective and antioxidant effect on 3T3 dermic fibroblast cells, treated with hydrogen peroxide and the scavenging capacity of free radicals was determined by the 2, 2-diphenyl-l-picrylhydrazyl (DPPH method as well as the Reducing Power of the ethanolic extracts of the leaves of the Machaerium floribundum. Minimal bactericidal concentrations (MBC were obtained against Propionibacterium acnes and Staphylococcus aureus of 5 mg/mL and 2 mg/mL, respectively. A cytoprotective effect of 111% was observed over the cellular viability of the fibroblasts at 10 μg/mL and an antioxidant effect of 92% over the viability of the fibroblasts treated with hydrogen peroxide at 25 μg/mL. A stimulation of 24% growth of fibroblasts at 50 μg/mL was evidenced. On the other hand a 93% scavenging activity of the DPPH free radical was shown for 100 μg/mL with a CI50 of 34 μg/mL. The reducing power was evidenced to be dependent on the concentration. The results obtained indicated that the ethanolic extract of Machaerium floribundum shows a good antibacterial activity against bacteria that induce acne and a high potential for scavenging of free radicals at relatively low concentrations.

  2. Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

    Directory of Open Access Journals (Sweden)

    Robinson Claire M

    2012-08-01

    Full Text Available Abstract Background Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu. As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. Methods CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR was used to examine the methylation status of the Thy-1 promoter. Results Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. Conclusion These data suggest that global and

  3. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

    Directory of Open Access Journals (Sweden)

    Christian Lacks Lino Cardenas

    Full Text Available As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF, remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls. In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that

  4. Poly(ADP-ribose) polymerase inhibition reduces tumor necrosis factor-induced inflammatory response in rheumatoid synovial fibroblasts

    NARCIS (Netherlands)

    García, S.; Bodaño, A.; Pablos, J. L.; Gómez-Reino, J. J.; Conde, C.

    2008-01-01

    To investigate the effect of poly(ADP-ribose) polymerase (PARP) inhibition on the production of inflammatory mediators and proliferation in tumour necrosis factor (TNF)-stimulated fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Cultured FLS from patients with RA were

  5. The hallmarks of fibroblast ageing.

    Science.gov (United States)

    Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

    2014-06-01

    Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Metabolic effects of basic fibroblast growth factor in streptozotocin-induced diabetic rats: A 1H NMR-based metabolomics investigation

    OpenAIRE

    Lin, Xiaodong; Zhao, Liangcai; Tang, Shengli; Zhou, Qi; Lin, Qiuting; Li, Xiaokun; Zheng, Hong; Gao, Hongchang

    2016-01-01

    The fibroblast growth factors (FGFs) family shows a great potential in the treatment of diabetes, but little attention is paid to basic FGF (bFGF). In this study, to explore the metabolic effects of bFGF on diabetes, metabolic changes in serum and feces were analyzed in the normal rats, the streptozocin (STZ)-induced diabetic rats and the bFGF-treated diabetic rats using a 1H nuclear magnetic resonance (NMR)-based metabolomic approach. Interestingly, bFGF treatment significantly decreased glu...

  7. Generation of a human induced pluripotent stem cell line (MUSIi001-A from caesarean section scar fibroblasts using Sendai viral vectors

    Directory of Open Access Journals (Sweden)

    Methichit Wattanapanitch

    2018-03-01

    Full Text Available We generated a human induced pluripotent stem cell (iPSC line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

  8. Molecular Insights into SIRT1 Protection Against UVB-Induced Skin Fibroblast Senescence by Suppression of Oxidative Stress and p53 Acetylation.

    Science.gov (United States)

    Chung, Ki Wung; Choi, Yeon Ja; Park, Min Hi; Jang, Eun Ji; Kim, Dae Hyun; Park, Byung Hyun; Yu, Byung Pal; Chung, Hae Young

    2015-08-01

    Stresses, such as exposure to ultraviolet radiation and those associated with aging, are known to cause premature cellular senescence that is characterized by growth arrest and morphological and gene expression changes. This study was designed to investigate the protective effect of Sirtuin1 (SIRT1) on the UVB-induced premature senescence. Under in vitro experimental conditions, exposure to a subcytotoxic dose of UVB enhanced human skin fibroblasts senescence, as characterized by increased β-galactosidase activity and increased levels of senescence-associated proteins. However, adenovirus-mediated SIRT1 overexpression significantly protected fibroblasts from UVB-induced cellular deterioration. Exposure to UVB-induced cell senescence was associated with oxidative stress and p38 mitogen-activated protein kinase activation. Molecular analysis demonstrated that deacetylation of Forkhead box O3α (FOXO3α) by SIRT1 changed the transcriptional activity of FOXO3α and increased resistance to the oxidative stress. In addition, SIRT1 suppressed UVB-induced p53 acetylation and its transcriptional activity, which directly affected the cell cycle arrest induced by UVB. Further study demonstrated that SIRT1 activation inhibited cell senescence in the skin of the HR1 hairless mouse exposed to UVB. The study identifies a new role for SIRT1 in the UVB-induced senescence of skin fibroblats and provides a potential target for skin protection through molecuar insights into the mechanisms responsible for UVB-induced photoaging. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts

    International Nuclear Information System (INIS)

    Crompton, N.E.A.; Emery, G.C.; Shi Yuquan; Sigg, M.; Blattmann, H.

    1998-01-01

    We investigated various phenotypic characteristics of radiation-induced morphologically transformed C3H 10T1/2 mouse fibroblasts. The cells were treated with 8 Gy x-rays, and type II/III foci were isolated. Cell lines were developed from these foci, and subsequently clones were established from these focal lines. The clones were examined for DNA content, radiosensitivity and inducible cell cycle arrests. Besides the morphological changes associated with the transformed state, the major difference between the isolated focal lines or derived clones and the parental C3H 10T1/2 line was one of ploidy. The transformed cells often displayed aneuploid and multiple polyploid populations. No change in the radiosensitivity of the transformed cells was observed. Furthermore, the two major radiation- and staurosporine-induced G1 and G2 cell cycle arrests observed in the parental cell line were also observed in the morphological transformants, suggesting that checkpoint function was normal. (orig.)

  10. The human PKP2/plakophilin-2 gene is induced by Wnt/β-catenin in normal and colon cancer-associated fibroblasts.

    Science.gov (United States)

    Niell, Núria; Larriba, María Jesús; Ferrer-Mayorga, Gemma; Sánchez-Pérez, Isabel; Cantero, Ramón; Real, Francisco X; Del Peso, Luis; Muñoz, Alberto; González-Sancho, José Manuel

    2018-02-15

    Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/β-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/β-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/β-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by β-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/β-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  11. Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1987-01-01

    We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H 2 O 2 , and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H 2 O 2 , the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein

  12. Cadmium induced changes in cell organelles: An ultrastructural study using cadmium sensitive and resistant muntjac fibroblast cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Ord, M.J.; Chibber, R.; Bouffler, S.D.

    1988-09-01

    A detailed electron microscopy study of cadmium sensitive and resistant muntjac fibroblast cell lines has identified a wide range of intracellular damage following exposure to cadmium. Damaged organelles included cell membrane, mitochondria, Golgi cisternae and tubular network, chromatin, nucleoli, microfilaments and ribosomes. Although cell membrane damage was generally the earliest indication of adverse cadmium action, particularly with continuous cadmium exposures, cells could tolerate extensive membrane loss. Mitochondrial distortion and some damage to Golgi was also tolerated. The turning point at which cadmium became lethal was generally marked by a cascade of events which included damage to both nuclear and cytoplasmic components. These results for fibroblasts are discussed and compared with damage reported in other types of cells.

  13. Plasma Rich in Growth Factors Inhibits Ultraviolet B Induced Photoageing of the Skin in Human Dermal Fibroblast Culture.

    Science.gov (United States)

    Anitua, Eduardo; Pino, Ander; Orive, Gorka

    Ultraviolet irradiation is able to deeply penetrate into the dermis and alter fibroblast structure and function, leading to a degradation of the dermal extracellular matrix. The regenerative effect of plasma rich in growth factors (PRGF) on skin ageing was investigated using UVB photo-stressed human dermal fibroblasts as an in vitro culture model. PRGF was assessed over the main indicative features of ultraviolet B irradiation, including ROS formation, cell viability and death detection, apoptosis/ necrosis analysis and biosynthetic activity measurement. Four different UV irradiation protocols were tested in order to analyze the beneficial effects of PRGF. Ultraviolet irradiation exhibited a dose dependent cytotoxicity and dose of 400mJ/cm2 was selected for subsequent experiments. PRGF increased the cell viability and decreased the cell death comparing to the non-treated group. The apoptosis and necrosis were significantly lower in PRGF treated fibroblasts. ROS production after UV irradiation was significantly reduced in the presence of PRGF. Procollagen type I, hyaluronic acid and TIMP-1 levels were higher in the when treated with PRGF. This preliminary in vitro study suggests that PRGF is able to prevent UVB derived photooxidative stress and to diminish the cell damage caused by ultraviolet irradiation.

  14. Interleukin 1β induces rapid phosphorylation and redistribution of talin: A possible mechanism for modulation of fibroblast focal adhesion

    International Nuclear Information System (INIS)

    Qwarnstroem, E.E.; MacFarlane, S.A.; Page, R.C.; Dower, S.K.

    1991-01-01

    The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1β/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1β and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1β incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [ 32 P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1β on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing

  15. Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

    Science.gov (United States)

    Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

    2010-06-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

  16. CopA3 Peptide Prevents Ultraviolet-Induced Inhibition of Type-I Procollagen and Induction of Matrix Metalloproteinase-1 in Human Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Dong-Hee Kim

    2014-05-01

    Full Text Available Ultraviolet (UV exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1 activity. A 9-mer peptide, CopA3 (CopA3 was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging.

  17. Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes.

    Science.gov (United States)

    Kuhn, Donald E; Roy, Sashwati; Radtke, Jared; Khanna, Savita; Sen, Chandan K

    2007-03-01

    Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.

  18. Hypoxia-induced autophagy is inhibited by PADI4 knockdown, which promotes apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

    Science.gov (United States)

    Fan, Tingting; Zhang, Changsong; Zong, Ming; Fan, Lieying

    2018-01-01

    Impaired apoptosis of rheumatoid arthritis (RA)-fibroblast-like synoviocytes (FLS) is pivotal in the process of RA. Peptidyl arginine deiminase type IV (PADI4) is associated with autoantibody regulation via histone citrullination in RA. The present study aimed to investigate the role of PADI4 in the apoptosis of RA-FLS. FLS were isolated from patients with RA and a rat model. The effects of PADI4 on RA-FLS were investigated in vitro and in vivo. Hypoxia-induced autophagy was induced by 1% O2 and was detected by immunohistochemical and immunofluorescence analysis; in addition, apoptosis was detected by flow cytometry. RA-FLS obtained from RA rat model exhibited significant proliferation under severe hypoxia conditions. Hypoxia also significantly induced autophagy and elevated the expression of PADI4. Subsequently, short hairpin RNA-mediated PADI4 knockdown was demonstrated to significantly inhibit hypoxia-induced autophagy and promote apoptosis in RA-FLS. The results of these in vitro and in vivo studies suggested that PADI4 may be closely associated with hypoxia-induced autophagy, and the inhibition of hypoxia-induced autophagy by PADI4 knockdown may contribute to an increase in the apoptosis of RA-FLS. PMID:29393388

  19. Chronic Exposure to Rhodobacter Sphaeroides Extract Lycogen™ Prevents UVA-Induced Malondialdehyde Accumulation and Procollagen I Down-Regulation in Human Dermal Fibroblasts

    Science.gov (United States)

    Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

    2014-01-01

    UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP)-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA) accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 μM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications. PMID:24463291

  20. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M

    2002-12-29

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to ({+-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydro= benzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody

  1. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    International Nuclear Information System (INIS)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M.

    2002-01-01

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not

  2. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    DEFF Research Database (Denmark)

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

  3. Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

    Science.gov (United States)

    Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

    2018-01-01

    Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

  4. Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2012-01-01

    Full Text Available In this study, we determined the molecular mechanism of γ-tocotrienol (GTT in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs. Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P<0.05. GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P<0.05. Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P<0.05 in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

  5. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    International Nuclear Information System (INIS)

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content ∼ 50-fold and their carboxypeptidase. A content ∼ 100-fold, and augment ∼ their biosynthesis of proteoglycans bearing 35 S-labeled haparin relative to 35 S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment

  6. Malignant transformation of human fibroblasts by neutrons and by gamma radiation: Relationship of mutations induced. Final report, January 1, 1993--December 31, 1995

    International Nuclear Information System (INIS)

    McCormick, J.J.

    1997-01-01

    The overall purpose of this project was to compare on a quantitative basis, the transforming and mutagenic effect of ionizing radiation ( 60 Co) and neutron radiation on human fibroblasts irradiated at different phases of the cell cycle. These studies were undertaken since mouse fibroblasts (C3H10T1/2) exhibit a window of sensitivity to ionizing radiation-induced transformation in late G2 and perhaps M and to neutron-induced transformation in G1 and in G2. In the 10T1/2 cells, essentially all the induced transformants come from cells that are in the sensitive windows at the time of irradiation. The mechanism responsible for the sensitive windows in the 10T1/2 cells has not yet been elucidated. Because of the aneuploid nature of 10T1/2 cells and the fact that mouse chromosomes are very small and nearly identical in size, some types of experiments that might indicate the mechanism are nearly impossible to carry out in these cells. Regardless of the mechanism, the fact that transformation results from a select subpopulation of cells has important practical implications for persons exposed to radiation. Whether a similar phenomena exists with other DNA damaging agents is not clear. Until this time, there has not been a human cell transformation assay that will allow one to quantitatively compare the transforming ability of radiation with different qualities. The MSU-1.1 cells had the potential to give such an assay. The authors developed this assay demonstrating a dose response and carrying out an extensive characterization of the focus-derived transformed cells including assays for tumorigenicity

  7. The expression change of β-arrestins in fibroblast-like synoviocytes from rats with collagen-induced arthritis and the effect of total glucosides of paeony.

    Science.gov (United States)

    Wang, Qing-Tong; Zhang, Ling-Ling; Wu, Hua-Xun; Wei, Wei

    2011-01-27

    To investigate the expression of β-arrestins in fibroblast-like synoviocytes (FLS) from collagen-induced arthritis (CIA) rats and the effect of total glucosides of paeony (TGP). TGP and glucosides of tripterygium wilfordii (GTW) were intragastriclly administrated to collagen-induced arthritis (CIA) rats after immunization. The secondary inflammatory reaction was evaluated by hind paw swelling, polyarthritis index and histopathological changes. Antibodies to type II collagen (CII) were determined by enzyme-linked immunosorbent assay (ELISA). Synoviocyte proliferations were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The expression of β-arrestins in synoviocytes from CIA rats was measured by western blot. The administration of TGP (25, 50, 100 mg/kg) depressed hind paw swelling and decreased the arthritis scores of CIA rats. TGP improved the pathologic manifestations of CIA. Serum anti-CII antibodies level increased significantly in CIA rats, while TGP had no effect on it. Fibroblast-like synoviocytes (FLS) proliferation was inhibited by TGP (50, 100 mg/kg). On d14, d28 after immunization, β-arrestins expression greatly up-regulated in synoviocytes from CIA rats and then returned to baseline levels on d42 after immunization. TGP (50, 100 mg/kg) significantly reduced the expression of β-arrestins. An inflammatory process in vivo induces an up-regulation of β-arrestins in synoviocytes from CIA rats while TGP can inhibit this change, which might be one of the important mechanisms for TGP to produce a marked therapeutic effect on RA. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  8. Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Germini, Diego; Rodighiero, Isabella [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Mirandola, Prisco [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); De Conto, Flora; Medici, Maria-Cristina [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy); Gatti, Rita [Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma (Italy); Chezzi, Carlo; Calderaro, Adriana [Department of Clinical and Experimental Medicine, University of Parma, Parma (Italy)

    2013-05-25

    Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promoting cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.

  9. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  10. A feeder- and xeno-free human induced pluripotent stem cell line obtained from primary human dermal fibroblasts with epigenetic repression of reprogramming factors expression: GPCCi001-A

    Directory of Open Access Journals (Sweden)

    Michał Stefan Lach

    2017-04-01

    Full Text Available The primary human dermal fibroblasts (PHDFs from breast cancer patient were obtained to generate the human induced pluripotent stem cell line GPCCi001-A via lentiviral transfection. Thus, a modified EF1a-hSTEMCCA-loxP with tetO operator which regulates transgene expression was used. This method takes advantage of epigenetic regulation of transcription and allows for stable silencing of the reprogramming factors in obtained hiPS cells. To increase the potential utility of hiPSCs for clinical applications, they were adapted to feeder- and xeno-free conditions. The pluripotency of GPCCi001-A cell line and ability to differentiate into three germ layers was confirmed.

  11. Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

    Directory of Open Access Journals (Sweden)

    Georg Kern

    Full Text Available Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506 induced TGF-β-like effects, manifested by increased expression of NAD(PH-oxidase 4 (Nox4, transgelin, tropomyosin 1, and procollagen α1(V mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V mRNA in tacrolimus-treated cells, but induced procollagen α1(V expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.

  12. Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

    Science.gov (United States)

    Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

    2018-03-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

  13. Salubrinal protects human skin fibroblasts against UVB-induced cell death by blocking endoplasmic reticulum (ER) stress and regulating calcium homeostasis.

    Science.gov (United States)

    Ji, Chao; Yang, Bo; Huang, Shu-Ying; Huang, Jin-Wen; Cheng, Bo

    2017-12-02

    The role of UVB in skin photo damages has been widely reported. Overexposure to UVB will induce severe DNA damages in epidermal cells and cause most cytotoxic symptoms. In the present study, we tested the potential activity of salubrinal, a selective inhibitor of Eukaryotic Initiation Factor 2 (eIF2) -alpha phosphatase, against UV-induced skin cell damages. We first exposed human fibroblasts to UVB radiation and evaluated the cytosolic Ca 2+ level as well as the induction of ER stress. We found that UVB radiation induced the depletion of ER Ca 2+ and increased the expression of ER stress marker including phosphorylated PERK, CHOP, and phosphorylated IRE1α. We then determined the effects of salubrinal in skin cell death induced by UVB radiation. We observed that cells pre-treated with salubrinal had a higher survival rate compared to cells treated with UVB alone. Pre-treatment with salubrinal successfully re-established the ER function and Ca 2+ homeostasis. Our results suggest that salubrinal can be a potential therapeutic agents used in preventing photoaging and photo damages. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    Science.gov (United States)

    Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

    2016-01-01

    Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. PMID:27621603

  15. Oxygen enhancement ratios for glutathione-deficient human fibroblasts determined from the frequency of radiation induced micronuclei

    International Nuclear Information System (INIS)

    Midander, J.

    1982-01-01

    The yield of micronuclei (MN) was determined to study the radiosensitizing effect of oxygen on three human fibroblast strains, characterized by genetically defined differences in their glutathione (GSH) level. Cells were irradiated in paired experiments with x-ray doses of 2.66 and 6.65 gy in their exponential growth phase in a monolayer under oxic and anoxic conditions. Results indicated a reduced oxygen effect for the GSH deficient cells, the reduction of o.e.r. being most pronounced in the case of GSHsup(-/-) cells, when it was close to unity. The o.e.r. value was intermediate for the GSHsup(+/-) in comparison with the two other cell strains. It is concluded that the data indicate a correlation between the cellular content of GSH and the oxygen enhancement of the formation of micronuclei after irradiation. (U.K.)

  16. Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

    Science.gov (United States)

    Ramachandra, Rashmi; Namburi, Ramesh B; Ortega-Martinez, Olga; Shi, Xiaofeng; Zaia, Joseph; Dupont, Sam T; Thorndyke, Michael C; Lindahl, Ulf; Spillmann, Dorothe

    2014-02-01

    Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

  17. Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced DNA synthesis.

    NARCIS (Netherlands)

    J.C.M. Zwetsloot; A.P. Barbeiro; W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1986-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V

  18. Infección congénita por citomegalovirus en recién nacidos del estado de San Luis Potosí, México Congenital cytomegalovirus infection in newborn infants from the state of San Luis Potosí, Mexico

    Directory of Open Access Journals (Sweden)

    Daniel E Noyola

    2011-12-01

    Full Text Available OBJETIVO: Determinar la prevalencia de infección congénita por citomegalovirus en recién nacidos participantes en el programa de tamiz neonatal de los Servicios de Salud de San Luis Potosí. MATERIAL Y MÉTODOS: Se evaluó la presencia de citomegalovirus en muestras de sangre almacenadas en papel filtro. RESULTADOS. Se detectó la presencia de citomegalovirus en 10 (0.68% de 1 457 muestras estudiadas. No se encontraron diferencias en las características de los recién nacidos con infección congénita en comparación con aquéllos sin infección. CONCLUSIONES: Es necesario concientizar a los profesionales de la salud sobre la prevalencia e impacto de la infección congénita por citomegalovirus.OBJECTIVE: To determine the prevalence of congenital cytomegalovirus infection in newborn infants included in the neonatal screening program coordinated by the State Health Services in San Luis Potosí. MATERIAL AND METHODS: We evaluated the presence of cytomegalovirus in blood samples stored in filter paper. RESULTS: Cytomegalovirus was detected in 10 (0.68% of the 1 457 samples included in the study. There were no differences in the characteristics of infants with congenital infection compared to those without infection. CONCLUSIONS: It is necessary to increase awareness of health professionals regarding the prevalence and impact of congenital cytomegalovirus infection.

  19. Are female daycare workers at greater risk of cytomegalovirus infection? A secondary data analysis of CMV seroprevalence between 2010 and 2013 in Hamburg, Germany

    Directory of Open Access Journals (Sweden)

    Stranzinger, Johanna

    2016-04-01

    Full Text Available Background: Close contact with asymptomatic children younger than three years is a risk factor for a primary cytomegalovirus (CMV infection. In pregnant women, such primary infection increases the risk of CMV-induced feto- or embryopathy. Daycare providers have therefore implemented working restrictions for pregnant daycare workers (DCWs in accordance with legislation and guidelines for maternity protection. However, little is known about the infection risk for DCWs. We therefore compared the prevalence of CMV antibodies of pregnant DCWs to that of female blood donors (BDs.Method: In a secondary data analysis, the prevalence of anti-CMV IgG among pregnant DCWs (N=509 in daycare centers (DCCs was compared to the prevalence of female first-time BDs (N=14,358 from the greater region of Hamburg, Germany. Data collection took place between 2010 and 2013. The influence of other risk factors such as age, pregnancies and place of residence was evaluated using logistic regression models. Results: The prevalence of CMV antibodies in pregnant DCWs was higher than in female BDs (54.6 vs 41.5%; OR 1.6; 95%CI 1.3–1.9. The subgroup of BDs who had given birth to at least one child and who lived in the city of Hamburg (N=2,591 had a prevalence of CMV antibodies similar to the prevalence in pregnant DCWs (53.9 vs 54.6%; OR 0.9; 95%CI 0.8–1.2. Age, pregnancy history and living in the center of Hamburg were risk factors for CMV infections.Conclusion: The comparison of pregnant DCWs to the best-matching subgroup of female first-time BDs with past pregnancies and living in the city of Hamburg does not indicate an elevated risk of CMV infection among DCWs. However, as two secondary data sets from convenience samples were used, a more detailed investigation of the risk factors other than place of residence, age and maternity was not possible. Therefore, the CMV infection risk in DCWs should be further studied by taking into consideration the potential preventive

  20. Genomic Programming of Human Neonatal Dendritic Cells in Congenital Systemic and In Vitro Cytomegalovirus Infection Reveal Plastic and Robust Immune Pathway Biology Responses

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    Widad Dantoft

    2017-09-01

    Full Text Available Neonates and especially premature infants are highly susceptible to infection but still can have a remarkable resilience that is poorly understood. The view that neonates have an incomplete or deficient immune system is changing. Human neonatal studies are challenging, and elucidating host protective responses and underlying cognate pathway biology, in the context of viral infection in early life, remains to be fully explored. In both resource rich and poor settings, human cytomegalovirus (HCMV is the most common cause of congenital infection. By using unbiased systems analyses of transcriptomic resources for HCMV neonatal infection, we find the systemic response of a preterm congenital HCMV infection, involves a focused IFN regulatory response associated with dendritic cells. Further analysis of transcriptional-programming of neonatal dendritic cells in response to HCMV infection in culture revealed an early dominant IFN-chemokine regulatory subnetworks, and at later times the plasticity of pathways implicated in cell-cycle control and lipid metabolism. Further, we identify previously unknown suppressed networks associated with infection, including a select group of GPCRs. Functional siRNA viral growth screen targeting 516-GPCRs and subsequent validation identified novel GPCR-dependent antiviral (ADORA1 and proviral (GPR146, RGS16, PTAFR, SCTR, GPR84, GPR85, NMUR2, FZ10, RDS, CCL17, and SORT1 roles. By contrast a gene family cluster of protocadherins is significantly differentially induced in neonatal cells, suggestive of possible immunomodulatory roles. Unexpectedly, programming responses of adult and neonatal dendritic cells, upon HCMV infection, demonstrated comparable quantitative and qualitative responses showing that functionally, neonatal dendritic cell are not overly compromised. However, a delay in responses of neonatal cells for IFN subnetworks in comparison with adult-derived cells are notable, suggestive of subtle plasticity

  1. Genomic Programming of Human Neonatal Dendritic Cells in Congenital Systemic and In Vitro Cytomegalovirus Infection Reveal Plastic and Robust Immune Pathway Biology Responses.

    Science.gov (United States)

    Dantoft, Widad; Martínez-Vicente, Pablo; Jafali, James; Pérez-Martínez, Lara; Martin, Kim; Kotzamanis, Konstantinos; Craigon, Marie; Auer, Manfred; Young, Neil T; Walsh, Paul; Marchant, Arnaud; Angulo, Ana; Forster, Thorsten; Ghazal, Peter

    2017-01-01

    Neonates and especially premature infants are highly susceptible to infection but still can have a remarkable resilience that is poorly understood. The view that neonates have an incomplete or deficient immune system is changing. Human neonatal studies are challenging, and elucidating host protective responses and underlying cognate pathway biology, in the context of viral infection in early life, remains to be fully explored. In both resource rich and poor settings, human cytomegalovirus (HCMV) is the most common cause of congenital infection. By using unbiased systems analyses of transcriptomic resources for HCMV neonatal infection, we find the systemic response of a preterm congenital HCMV infection, involves a focused IFN regulatory response associated with dendritic cells. Further analysis of transcriptional-programming of neonatal dendritic cells in response to HCMV infection in culture revealed an early dominant IFN-chemokine regulatory subnetworks, and at later times the plasticity of pathways implicated in cell-cycle control and lipid metabolism. Further, we identify previously unknown suppressed networks associated with infection, including a select group of GPCRs. Functional siRNA viral growth screen targeting 516-GPCRs and subsequent validation identified novel GPCR-dependent antiviral (ADORA1) and proviral (GPR146, RGS16, PTAFR, SCTR, GPR84, GPR85, NMUR2, FZ10, RDS, CCL17, and SORT1) roles. By contrast a gene family cluster of protocadherins is significantly differentially induced in neonatal cells, suggestive of possible immunomodulatory roles. Unexpectedly, programming responses of adult and neonatal dendritic cells, upon HCMV infection, demonstrated comparable quantitative and qualitative responses showing that functionally, neonatal dendritic cell are not overly compromised. However, a delay in responses of neonatal cells for IFN subnetworks in comparison with adult-derived cells are notable, suggestive of subtle plasticity differences. These

  2. HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models.

    Science.gov (United States)

    Huh, Yun Hyun; Lee, Gyuseok; Lee, Keun-Bae; Koh, Jeong-Tae; Chun, Jang-Soo; Ryu, Je-Hwang

    2015-10-29

    Pannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion. Collagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis. HIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer. Our findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.

  3. Cyclophilin A secreted from fibroblast-like synoviocytes is involved in the induction of CD147 expression in macrophages of mice with collagen-induced arthritis

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    Nishioku Tsuyoshi

    2012-11-01

    Full Text Available Abstract Background Cyclophilin A (CypA, a member of the immunophilin family, is a ubiquitously distributed intracellular protein. Recent studies have shown that CypA is secreted by cells in response to inflammatory stimuli. Elevated levels of extracellular CypA and its receptor, CD147 have been detected in the synovium of patients with RA. However, the precise process of interaction between CypA and CD147 in the development of RA remains unclear. This study aimed to investigate CypA secretion from fibroblast-like synoviocytes (FLS isolated from mice with collagen-induced arthritis (CIA and CypA-induced CD147 expression in mouse macrophages. Findings CIA was induced by immunization with type II collagen in mice. The expression and localization of CypA and CD147 was investigated by immunoblotting and immunostaining. Both CypA and CD147 were highly expressed in the joints of CIA mice. CD147 was expressed in the infiltrated macrophages in the synovium of CIA mice. In vitro, spontaneous CypA secretion from FLS was detected and this secretion was increased by stimulation with lipopolysaccharide. CypA markedly increased CD147 levels in macrophages. Conclusions These findings suggest that an interaction in the synovial joints between extracellular CypA and CD147 expressed by macrophages may be involved in the mechanisms underlying the development of arthritis.

  4. Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

    International Nuclear Information System (INIS)

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-01-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. The gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives

  5. Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

    International Nuclear Information System (INIS)

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-01-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G 2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or #betta#-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G 2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H 2 O 2 , or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G 2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives

  6. H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Zhao, Shanmin; Li, Li; Wang, Shiyong; Yu, Chenlin; Xiao, Bang; Lin, Lifang; Cong, Wei; Cheng, Jishuai; Yang, Wenjing; Sun, Wei; Cui, Shufang

    2016-12-20

    Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

  7. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Yoshino A

    2016-08-01

    Full Text Available Atsushi Yoshino,1 Natalia Polouliakh,1–3 Akira Meguro,1 Masaki Takeuchi,1,4 Tatsukata Kawagoe,1 Nobuhisa Mizuki1 1Department of Ophthalmology and Visual Science, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 2Sony Computer Science Laboratories Inc., Fundamental Research Laboratories, 3Systems Biology Institute, Tokyo, Japan; 4Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Abstract: Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. Keywords: fish egg, antiaging, gene expression analysis, antioxidative gene, phylogenetic footprinting analysis

  8. PDK4 Deficiency Induces Intrinsic Apoptosis in Response to Starvation in Fibroblasts from Doberman Pinschers with Dilated Cardiomyopathy.

    Science.gov (United States)

    Taggart, Kathryn; Estrada, Amara; Thompson, Patrick; Lourenco, Francisco; Kirmani, Sara; Suzuki-Hatano, Silveli; Pacak, Christina A

    2017-01-01

    The Doberman pinscher (DP) canine breed displays a high incidence of idiopathic, nonischemic dilated cardiomyopathy (DCM) with increased mortality. A common mutation in DPs is a splice site deletion in the pyruvate dehydrogenase kinase 4 (PDK4) gene that shows a positive correlation with DCM development. PDK4, a vital mitochondrial protein, controls the switch between glycolysis and oxidative phosphorylation based upon nutrient availability. It is likely, although unproven, that DPs with the PDK4 mutation are unable to switch to oxidative phosphorylation during periods of low nutrient availability, and thus are highly susceptible to mitochondrial-mediated apoptosis. This study investigated cell viability, mitochondrial stress, and activation of the intrinsic (mitochondrial mediated) apoptotic pathway in dermal fibroblasts from DPs that were healthy (PDK4 wt/wt ), heterozygous (PDK4 wt/del ), and homozygous (PDK4 del/del ) for the PDK4 mutation under conditions of high (unstarved) and low (starved) nutrient availability in vitro . As hypothesized, PDK4 wt/del and PDK4 del/del cells showed evidence of mitochondrial stress and activation of the intrinsic apoptotic pathway following starvation, while the PDK4 wt/wt cells remained healthy and viable under these conditions. Adeno-associated virus (AAV) PDK4-mediated gene replacement experiments confirmed cause-effect relationships between PDK4 deficiency and apoptosis activation. The restoration of function observed following administration of AAV-PDK4 provides strong support for the translation of this gene therapy approach into the clinical realm for PDK4-affected Dobermans.

  9. CD147 promotes IKK/IκB/NF-κB pathway to resist TNF-induced apoptosis in rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Zhai, Yue; Wu, Bo; Li, Jia; Yao, Xi-ying; Zhu, Ping; Chen, Zhi-nan

    2016-01-01

    TNF is highly expressed in synovial tissue of rheumatoid arthritis (RA) patients, where it induces proinflammatory cytokine secretion. However, in other cases, TNF will cause cell death. Considering the abnormal proliferation and activation of rheumatoid arthritis synovioblasts, the proper rate of synovioblast apoptosis could possibly relieve arthritis. However, the mechanism mediating TNF-induced synovioblast survival versus cell death in RA is not fully understood. Our objective was to study the role of CD147 in TNF downstream pathway preference in RA synovioblasts. We found that overexpressing TNF in synovial tissue did not increase the apoptotic level and, in vitro, TNF-induced mild synovioblast apoptosis and promoted IL-6 secretion. CD147, which was highly expressed in rheumatoid arthritis synovial fibroblasts (RASFs), increased the resistance of synovioblasts to apoptosis under TNF stimulation. Downregulating CD147 both increased the apoptotic rate and inhibited IκB kinase (IKK)/IκB/NF-κB pathway-dependent proinflammatory cytokine secretion. Further, we determined that it was the extracellular portion of CD147 and not the intracellular portion that was responsible for synovioblast apoptosis resistance. CD147 monoclonal antibody inhibited TNF-induced proinflammatory cytokine production but had no effect on apoptotic rates. Thus, our study indicates that CD147 is resistant to TNF-induced apoptosis by promoting IKK/IκB/NF-κB pathway, and the extracellular portion of CD147 is the functional region. CD147 inhibits TNF-stimulated RASF apoptosis. CD147 knockdown decreases IKK expression and inhibits NF-κB-related cytokine secretion. CD147's extracellular portion is responsible for apoptosis resistance. CD147 antibody inhibits TNF-related cytokine secretion without additional apoptosis.

  10. Fibroblast growth factor-2-induced host stroma reaction during initial tumor growth promotes progression of mouse melanoma via vascular endothelial growth factor A-dependent neovascularization.

    Science.gov (United States)

    Tsunoda, Satoshi; Nakamura, Toshiyuki; Sakurai, Hiroaki; Saiki, Ikuo

    2007-04-01

    Fibroblast growth factor (FGF)-2 has been considered to play a critical role in neovascularization in several tumors; however, its precise role in tumor progression is not fully understood. In the present study, we have characterized the role of FGF-2 in B16-BL6 mouse melanoma cells, focusing on effects during the initial phase of tumor growth. FGF-2 was injected at the tumor inoculation site of dorsal skin during the initial phase. FGF-2 induced marked tumor growth and lymph node metastasis. This was well correlated with an increase in neovascularization in the host stroma. FGF-2 also recruited inflammatory and mesenchymal cells in host stroma. Marked tumor growth, pulmonary metastasis and intensive neovascularization in tumor parenchyma were also observed after a single injection of FGF-2 into the footpad inoculation site. In contrast, repeated injections of FGF-2 at a site remote from the footpad tumor were ineffective in promoting tumor growth and metastasis. These promoting activities of FGF-2 were blocked by local injections of a glucocorticoid hormone, suggesting that host inflammatory responses induced by FGF-2 are associated with FGF-2-induced tumor progression. In addition, although FGF-2 did not promote cellular proliferation and vascular endothelial growth factor A (VEGFA) mRNA expression in B16-BL6 cells in vitro, FGF-2 induced VEGFA expression in host stroma rather than tumor tissue, and local injections of a neutralizing antibody against VEGFA inhibited these activities of FGF-2 in vivo. These results indicate that abundant FGF-2 during the initial phase of tumor growth induces VEGFA-dependent intensive neovascularization in host stroma, and supports marked tumor growth and metastasis.

  11. Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.

    Science.gov (United States)

    Luo, Limin; Li, Jun; Liu, Han; Jian, Xiaoqing; Zou, Qianlei; Zhao, Qing; Le, Qu; Chen, Hongdou; Gao, Xinghua; He, Chundi

    2017-05-12

    Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those

  12. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Hsin-Yu Chou

    2016-06-01

    Full Text Available Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA, and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR, we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

  13. Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

    Science.gov (United States)

    Yoo, T; Ham, S A; Hwang, J S; Lee, W J; Paek, K S; Oh, J W; Kim, J H; Do, J T; Han, C W; Kim, J H; Seo, H G

    2016-10-01

    We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

    Science.gov (United States)

    Uboldi, Chiara; Urbán, Patricia; Gilliland, Douglas; Bajak, Edyta; Valsami-Jones, Eugenia; Ponti, Jessica; Rossi, François

    2016-03-01

    The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. PDK4 Deficiency Induces Intrinsic Apoptosis in Response to Starvation in Fibroblasts from Doberman Pinschers with Dilated Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Kathryn Taggart

    2017-12-01

    Full Text Available The Doberman pinscher (DP canine breed displays a high incidence of idiopathic, nonischemic dilated cardiomyopathy (DCM with increased mortality. A common mutation in DPs is a splice site deletion in the pyruvate dehydrogenase kinase 4 (PDK4 gene that shows a positive correlation with DCM development. PDK4, a vital mitochondrial protein, controls the switch between glycolysis and oxidative phosphorylation based upon nutrient availability. It is likely, although unproven, that DPs with the PDK4 mutation are unable to switch to oxidative phosphorylation during periods of low nutrient availability, and thus are highly susceptible to mitochondrial-mediated apoptosis. This study investigated cell viability, mitochondrial stress, and activation of the intrinsic (mitochondrial mediated apoptotic pathway in dermal fibroblasts from DPs that were healthy (PDK4wt/wt, heterozygous (PDK4wt/del, and homozygous (PDK4del/del for the PDK4 mutation under conditions of high (unstarved and low (starved nutrient availability in vitro. As hypothesized, PDK4wt/del and PDK4del/del cells showed evidence of mitochondrial stress and activation of the intrinsic apoptotic pathway following starvation, while the PDK4wt/wt cells remained healthy and viable under these conditions. Adeno-associated virus (AAV PDK4-mediated gene replacement experiments confirmed cause-effect relationships between PDK4 deficiency and apoptosis activation. The restoration of function observed following administration of AAV-PDK4 provides strong support for the translation of this gene therapy approach into the clinical realm for PDK4-affected Dobermans.

  16. Cytomegalovirus Infections among African-Americans

    Directory of Open Access Journals (Sweden)

    Best Al M

    2008-08-01

    Full Text Available Abstract Background Since African-Americans have twice the prevalence of cytomegalovirus (CMV infections as age-matched Caucasians we sought to determine the ages and possible sources of infection of African-American children. Methods Subjects were 157 African-American healthy children and adolescents and their 113 household adults in Richmond VA. Families completed a questionnaire, provided saliva for antibody testing, and adolescents were interviewed regarding sexual activity. Results Regardless of age CMV seropositivity was not associated with gender, breast feeding, health insurance, sexual activity, or household income, education, or size. In the final regression model, prior CMV infection in adults was over two-fold higher than in children (chi-square = 18.8, p Conclusion We observed that African-American children had CMV seroprevalence rates by age 20 years at less than one-half of that of their adult mothers and caregivers. Sibling-to-sibling transmission was a likely source of CMV infections for the children. The next generation of African-American women may be highly susceptible to a primary CMV infection during pregnancy and may benefit from a CMV vaccine.

  17. seroprevalence of cytomegalovirus infection amongst pregnant

    African Journals Online (AJOL)

    boaz

    Cytomegalovirus (CMV) is a major public health problem throughout the world. It is the leading cause of ... Serum obtained from the blood samples were examined ... systems have been weakened by disease or drug ... fluids (e.g. saliva, urine, breast milk cervico-vaginal ... centrifuged on same day and the serum stored at -.

  18. Attitudes toward newborn screening for cytomegalovirus infection.

    Science.gov (United States)

    Din, Erica S; Brown, Cedric J; Grosse, Scott D; Wang, Chengbin; Bialek, Stephanie R; Ross, Danielle S; Cannon, Michael J

    2011-12-01

    Newborns are not routinely screened for cytomegalovirus (CMV), the leading infectious cause of developmental disability. Congenital CMV satisfies a number of criteria for inclusion in newborn screening, and screening potentially offers benefits. Screening could also introduce harms such as anxiety and unnecessary costs for the families of the substantial proportion of CMV-infected children who never develop CMV-related disabilities. Our objective was to assess attitudes toward newborn screening for CMV. We analyzed responses to 5 statements about CMV and newborn screening from 3922 participants in the 2009 HealthStyles survey, a national mail survey designed to include a group similar to the US population with respect to gender, age, race/ethnicity, income, and household size. Two-step cluster analysis was performed to identify clusters of parental attitudes. The majority of respondents strongly or somewhat agreed that they would want to have their newborn tested for CMV even if it was not performed routinely (84%), they had to pay $20 (87%), or CMV-related problems never developed (84%). Nearly half (47%) of them "would worry that the CMV test would lead to unneeded doctor visits and expenses," and 32% "think CMV problems are too rare to worry about." Three clusters of parent respondents were identified on the basis of their attitudes toward CMV screening: "strongly in favor" (31%), "moderately in favor" (49%), and "weakly opposed" (20%). Among most parents, costs, worry, and anxiety associated with newborn screening for CMV would be acceptable. Although attitudes were generally favorable, a minority of the parents were weakly opposed to newborn screening for CMV.

  19. Prenatal Diagnosis of Congenital Cytomegalovirus Infection

    Science.gov (United States)

    Lazzarotto, T.; Guerra, B.; Spezzacatena, P.; Varani, S.; Gabrielli, L.; Pradelli, P.; Rumpianesi, F.; Banzi, C.; Bovicelli, L.; Landini, M. P.

    1998-01-01

    We report here the results of a study on the prenatal diagnosis of congenital cytomegalovirus (CMV) infection. The study was carried out by both PCR and virus isolation from amniotic fluid (AF) for 82 pregnant women at risk of transmitting CMV for the detection of (i) seroconversion to CMV immunoglobulin G (IgG) positivity during the first trimester of pregnancy, (ii) symptomatic CMV infection in the mother during the first trimester of pregnancy or intrauterine growth retardation detected by ultrasound or abnormal ultrasonographic findings suggestive of fetal infections, and (iii) seropositivity for CMV-specific IgM. For 50 women, fetal blood (FB) was also obtained and tests for antigenemia and PCR were performed. The results indicate that AF is better than FB for the prenatal diagnosis of CMV infection. PCR with AF has a sensitivity (SNS) of 100%, a specificity (SPE) of 83.3%, a positive predictive value (PPV) of 40%, and a negative predictive value (NPV) of 100%; rapid virus isolation with the same material has an SNS of 50%, an SPE of 100%, a PPV of 100%, and an NPV of 94.7%. Fewer than 10% of the women positive for IgM by enzyme immunoassay (EIA) had a congenitally infected fetus or newborn infant. When EIA IgM positivity was confirmed by Western blotting (WB) and the WB profile was considered, the percent transmission detected among women with an “at-risk” profile was higher than that observed among IgM-positive women and was the same as that among women who seroconverted during the first trimester of pregnancy (transmission rates of 29 and 25%, respectively). PMID:9817869

  20. Seroepidemiology of cytomegalovirus infections in Croatia.

    Science.gov (United States)

    Vilibic-Cavlek, Tatjana; Kolaric, Branko; Beader, Natasa; Vrtar, Izabela; Tabain, Irena; Mlinaric-Galinovic, Gordana

    2017-02-01

    Cytomegalovirus (CMV) is endemic worldwide, with marked differences in the seroprevalence rates between countries. The aim of this study was to analyze the seroprevalence of CMV infections in Croatia. During a 3-year period (2013-2015) 2438 consecutive serum samples collected from Croatian residents were tested for the presence of CMV IgM and IgG antibodies using enzyme-linked immunoassay. The IgM/IgG positive samples were further tested for IgG avidity. The overall seroprevalence rates for CMV IgG and IgM antibodies were 74.4 % and 4.3 %, respectively. The IgG seroprevalence showed significant differences between population groups: children/adolescents 54.6 %, general adult population 77.2 %, hemodialysis patients 91.4 % (p < 0.001). Seropositivity of CMV was strongly age-dependent with prevalences ranging from 53.0 % in children less than 10 years old to 93.8 % in persons above 60 years (p < 0.001). There was no difference in the prevalence rate between women with normal pregnancy and women with poor obstetric history. Gender and place of residence were not associated with CMV seropositivity. Using IgG avidity, current/recent primary CMV infection was confirmed by a low/borderline avidity index (AI) in 46.7 % participants, while in 53.3 % a high AI indicated CMV reactivation or reinfection. Primary infections were detected mainly in children and adolescents (83.2 % and 70.5 %, respectively), while reactivation/reinfection was common in persons older than 40 (77.0-100 %). Reactivation/reinfection was most commonly detected in hemodialysis patients (92.3 %). Logistic regression showed that older age and being on hemodialysis were significant predictors of CMV seropositivity. Infections with CMV are widespread in the Croatian population. Older age and being on hemodialysis appear to be the main risk factors for CMV infection.

  1. Human cytomegalovirus infections in premature infants by ...

    African Journals Online (AJOL)

    Freezing breast milk may be protective for the preterm infant until the titer of CMV antibody increases. However clinical importance of CMV infection in premature infants by breast-feeding is still unclear. This minireview focuses on recent advances in the study of CMV infection in premature infants by breastfeeding.

  2. Advances in congenital and postnatal cytomegalovirus infections

    NARCIS (Netherlands)

    Gunkel, J.

    2017-01-01

    Congenital CMV infection (cCMV) is the most prevalent viral infection worldwide and the leading cause of non-genetic sensorineural hearing loss. Early diagnosis of cCMV infection is advantageous as it allows for regular follow-up and timely intervention in case of late-onset symptoms among

  3. Postnatally acquired cytomegalovirus infections in preterm infants

    NARCIS (Netherlands)

    Nijman, J.

    2013-01-01

    A postnatal cytomegalovirus (CMV) infection is common in very low birth weight infants with an estimated prevalence of 6–59%. Breast milk from CMV seropositive mothers is the main source of postnatal CMV infection. Ninety-six percent of these mothers shed CMV in their breast milk after delivery due

  4. Strongyloides Hyperinfection Syndrome Combined with Cytomegalovirus Infection

    Directory of Open Access Journals (Sweden)

    Fatehi Elnour Elzein

    2016-01-01

    Full Text Available The mortality in Strongyloides hyperinfection syndrome (SHS is alarmingly high. This is particularly common in bone marrow, renal, and other solid organ transplant (SOT patients, where figures may reach up to 50–85%. Immunosuppressives, principally corticosteroids, are the primary triggering factor. In general, the clinical features of Strongyloides stercoralis hyperinfection are nonspecific; therefore, a high index of suspicion is required for early diagnosis and starting appropriate therapy. Although recurrent Gram-negative sepsis and meningitis have been previously reported, the combination of both cytomegalovirus (CMV and strongyloidiasis had rarely been associated. We here describe a patient who survived SHS with recurrent Escherichia coli (E. coli urosepsis and CMV infection.

  5. No. 240-Cytomegalovirus Infection in Pregnancy.

    Science.gov (United States)

    Yinon, Yoav; Farine, Dan; Yudin, Mark H

    2018-02-01

    To review the principles of prenatal diagnosis of congenital cytomegalovirus (CMV) infection and to describe the outcomes of the affected pregnancies. Effective management of fetal infection following primary and secondary maternal CMV infection during pregnancy. Neonatal signs include intrauterine growth restriction (IUGR), microcephaly, hepatosplenomegaly, petechiae, jaundice, chorioretinitis, thrombocytopenia and anemia, and long-term sequelae consist of sensorineural hearing loss, mental retardation, delay of psychomotor development, and visual impairment. These guidelines provide a framework for diagnosis and management of suspected CMV infections. Medline was searched for articles published in English from 1966 to 2009, using appropriate controlled vocabulary (congenital CMV infection) and key words (intrauterine growth restriction, microcephaly). Results were restricted to systematic reviews, randomized controlled trials/controlled clinical trials, and observational studies. Searches were updated on a regular basis and incorporated into the guideline. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. The quality of evidence reported in this document has been assessed using the evaluation of evidence criteria in the Report of the Canadian Task Force on Preventive Health Care (Table 1). Copyright © 2018. Published by Elsevier Inc.

  6. Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression

    Directory of Open Access Journals (Sweden)

    Hoe-Yune Jung

    2014-01-01

    Full Text Available Exposure to ultraviolet (UV light can cause skin photoaging, which is associated with upregulation of matrix metalloproteinases (MMPs and downregulation of collagen synthesis. It has been reported that MMPs, especially MMP-1, MMP-3 and MMP-9, decrease the elasticity of the dermis by degrading collagen. In this study, we assessed the effects of Pinus densiflora extract (PDE on photoaging and investigated its mechanism of action in human skin fibroblast (Hs68 cells after UVB exposure using real-time polymerase chain reaction, Western blot analysis, and enzymatic activity assays. PDE exhibited an antioxidant activity and inhibited elastase activities in vitro. We also found that PDE inhibited UVB-induced cytotoxicity, MMP-1 production and expression of MMP-1, -3 and -9 mRNA in Hs68 cells. In addition, PDE decreased UVB-induced MMP-2 activity and MMP-2 mRNA expression. Moreover, PDE prevented the decrease of type I procollagen mediated by exposure to UVB irradiation, an effect that is linked to the upregulation and downregulation of Smad3 and Smad7, respectively. Another effect of UV irradiation is to stimulate activator protein 1 (AP-1 activity via overexpression of c-Jun/c-Fos, which, in turn, upregulates MMP-1, -3, and -9. In this study, we found that PDE suppressed UV-induced c-Jun and c-Fos mRNA expression. Taken together, these results demonstrate that PDE regulates UVB-induced expression of MMPs and type I procollagen synthesis by inhibiting AP-1 activity and restoring impaired Smad signaling, suggesting that PDE may be useful as an effective anti-photoaging agent.

  7. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. I. Pulsed-field gel electrophoresis method

    International Nuclear Information System (INIS)

    Rydberg, B.; Loebrich, M.; Cooper, P.K.

    1994-01-01

    The relative effectiveness of high-energy neon and iron ions for the production of DNA double-strand breaks was measured in one transformed and one nontransformed human fibroblast cell line using pulsed-field gel electrophoresis. The DNA released from the gel plug (fraction of activity released: FAR) as well as the size distribution of the DNA entering the gel were used to compare the effects of the heavy-ion exposure with X-ray exposure. Both methods gave similar results, indicating similar distributions of breaks over megabase-pair distances for the heavy ions and the X rays. The relative biological effectiveness (RBE) compared to 225 kVp X rays of initially induced DNA double-strand breaks was found to be 0.85 for 425 MeV/u neon ions (LET 32 keV/μm) and 0.42-0.55 for 250-600 MeV/u iron ions (LET 190-350 keV/μm). Postirradiation incubation showed less efficient repair of breaks induced by the neon ions and the 600 MeV/u iron ions compared to X rays. Survival experiments demonstrated RBE values larger than one for cell killing by the heavy ions in parallel experiments (neon: RBE = 1.2, iron: RBE = 2.3-3.0, based on D 10 values). It is concluded that either the initial yield of DNA double-strand breaks induced by the high-energy particles is lower than the yield for X rays, or the breaks induced by heavy ions are present in clusters that cannot be resolved with the technique used. These results are confirmed in the accompanying paper. 48 refs., 5 figs., 2 tabs

  8. Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9.

    Science.gov (United States)

    Yang, Yuanyuan; Zhang, Xiaobai; Yi, Li; Hou, Zhenzhen; Chen, Jiayu; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Sun, Xiao-Fang; Jiang, Cizhong; Wang, Yixuan; Gao, Shaorong

    2016-01-01

    Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. In the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with β-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as β-thalassemia. ©AlphaMed Press.

  9. Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

    Directory of Open Access Journals (Sweden)

    Líbalová Helena

    2012-01-01

    Full Text Available Abstract Background Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5 collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM from ambient air particles, human embryonic lung fibroblasts (HEL12469 were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. Method For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina. The expression of selected genes was verified by quantitative real-time PCR. Results Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair, the Wnt and TGF-β signaling pathways

  10. Flavonoids Derived from Abelmoschus esculentus Attenuates UV-B Induced Cell Damage in Human Dermal Fibroblasts Through Nrf2-ARE Pathway.

    Science.gov (United States)

    Patwardhan, Juilee; Bhatt, Purvi

    2016-05-01

    Ultraviolet-B (UV-B) radiation is a smaller fraction of the total radiation reaching the Earth but leads to extensive damage to the deoxyribonucleic acid (DNA) and other biomolecules through formation of free radicals altering redox homeostasis of the cell. Abelmoschus esculentus (okra) has been known in Ayurveda as antidiabetic, hypolipidemic, demulscent, antispasmodic, diuretic, purgative, etc. The aim of this study is to evaluate the protective effect of flavonoids from A. esculentus against UV-B-induced cell damage in human dermal fibroblasts. UV-B protective activity of ethyl acetate (EA) fraction of okra was studied against UV-B-induced cytotoxicity, antioxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. Flavonoid-rich EA fraction depicted a significant antioxidant potential also showing presence of rutin. Pretreatment of cells with EA fraction (10-30 μg/ml) prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Our study demonstrated for the 1(st) time that EA fraction of okra may reduce oxidative stress through Nrf2-ARE pathway as well as through endogenous enzymatic antioxidant system. These results suggested that flavonoids from okra may be considered as potential UV-B protective agents and may also be formulated into herbal sunscreen for topical application. Flavonoid-enriched ethyl acetate (EA) fraction from A. esculentus protected against ultraviolet-B (UV-B)-induced oxidative DNA damageEA fraction prevented UV-B-induced cytotoxicity, depletion of endogenous enzymatic antioxidants, and intracellular reactive oxygen species productionEA fraction could reduce oxidative stress through the Nrf2-ARE

  11. Oleic acid blocks EGF-induced [Ca2+]i release without altering cellular metabolism in fibroblast EGFR T17.

    Science.gov (United States)

    Zugaza, J L; Casabiell, X A; Bokser, L; Casanueva, F F

    1995-02-06

    EGFR-T17 cells were pretreated with oleic acid and 5-10 minutes later stimulated with EGF, to study if early ionic signals are instrumental in inducing metabolic cellular response. Oleic acid blocks EGF-induced [Ca2+]i rise and Ca2+ influx without altering 2-deoxyglucose and 2-aminobutiryc acid uptake nor acute, nor chronically. Oleic acid it is shown, in the first minutes favors the entrance of both molecules to modify the physico-chemical membrane state. On the other hand, oleic acid is unable to block protein synthesis. The results suggest that EGF-induced Ins(1,4,5)P3/Ca2+ pathway does not seem to be decisive in the control of cellular metabolic activity.

  12. Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

    Energy Technology Data Exchange (ETDEWEB)

    Karpus, Olga N.; Hsiao, Cheng-Chih; Kort, Hanneke de; Tak, Paul P.; Hamann, Jörg, E-mail: j.hamann@amc.uva.nl

    2016-08-26

    Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS. - Highlights: • Intracellular poly(I:C) and 3pRNA evoke immune responses in FLS. • Only intracellular delivery of poly(I:C) induces FLS apoptosis. • FLS do not require MDA5 for their response to intracellular poly(I:C). • FLS respond to intracellular poly(I:C) independent of IPS and STING. • An unknown intracellular sensor recognizes linear dsRNA in FLS.

  13. Dietary Manipulations That Induce Ketosis Activate the HPA Axis in Male Rats and Mice: A Potential Role for Fibroblast Growth Factor-21.

    Science.gov (United States)

    Ryan, Karen K; Packard, Amy E B; Larson, Karlton R; Stout, Jayna; Fourman, Sarah M; Thompson, Abigail M K; Ludwick, Kristen; Habegger, Kirk M; Stemmer, Kerstin; Itoh, Nobuyuki; Perez-Tilve, Diego; Tschöp, Matthias H; Seeley, Randy J; Ulrich-Lai, Yvonne M

    2018-01-01

    In response to an acute threat to homeostasis or well-being, the hypothalamic-pituitary-adrenocortical (HPA) axis is engaged. A major outcome of this HPA axis activation is the mobilization of stored energy, to fuel an appropriate behavioral and/or physiological response to the perceived threat. Importantly, the extent of HPA axis activity is thought to be modulated by an individual's nutritional environment. In this study, we report that nutritional manipulations signaling a relative depletion of dietary carbohydrates, thereby inducing nutritional ketosis, acutely and chronically activate the HPA axis. Male rats and mice maintained on a low-carbohydrate high-fat ketogenic diet (KD) exhibited canonical markers of chronic stress, including increased basal and stress-evoked plasma corticosterone, increased adrenal sensitivity to adrenocorticotropin hormone, increased stress-evoked c-Fos immunolabeling in the paraventricular nucleus of the hypothalamus, and thymic atrophy, an indicator of chronic glucocorticoid exposure. Moreover, acutely feeding medium-chain triglycerides (MCTs) to rapidly induce ketosis among chow-fed male rats and mice also acutely increased HPA axis activity. Lastly, and consistent with a growing literature that characterizes the hepatokine fibroblast growth factor-21 (FGF21) as both a marker of the ketotic state and as a key metabolic stress hormone, the HPA response to both KD and MCTs was significantly blunted among mice lacking FGF21. We conclude that dietary manipulations that induce ketosis lead to increased HPA axis tone, and that the hepatokine FGF21 may play an important role to facilitate this effect. Copyright © 2018 Endocrine Society.

  14. Fibroblast growth factor 21 protects mouse brain against D-galactose induced aging via suppression of oxidative stress response and advanced glycation end products formation.

    Science.gov (United States)

    Yu, Yinhang; Bai, Fuliang; Wang, Wenfei; Liu, Yaonan; Yuan, Qingyan; Qu, Susu; Zhang, Tong; Tian, Guiyou; Li, Siming; Li, Deshan; Ren, Guiping

    2015-06-01

    Fibroblast growth factor 21 (FGF21) is a hormone secreted predominantly in the liver, pancreas and adipose tissue. Recently, it has been reported that FGF21-Transgenic mice can extend their lifespan compared with wild type counterparts. Thus, we hypothesize that FGF21 may play some roles in aging of organisms. In this study d-galactose (d-gal)-induced aging mice were used to study the mechanism that FGF21 protects mice from aging. The three-month-old Kunming mice were subcutaneously injected with d-gal (180mg·kg(-1)·d(-1)) for 8weeks and administered simultaneously with FGF21 (1, 2 or 5mg·kg(-1)·d(-1)). Our results showed that administration of FGF21 significantly improved behavioral performance of d-gal-treated mice in water maze task and step-down test, reduced brain cell damage in the hippocampus, and attenuated the d-gal-induced production of MDA, ROS and advanced glycation end products (AGEs). At the same time, FGF21 also markedly renewed the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total anti-oxidation capability (T-AOC), and decreased the enhanced total cholinesterase (TChE) activity in the brain of d-gal-treated mice. The expression of aldose reductase (AR), sorbitol dehydrogenase (SDH) and member-anchored receptor for AGEs (RAGE) declined significantly after FGF21 treatment. Furthermore, FGF21 suppressed inflamm-aging by inhibiting IκBα degradation and NF-κB p65 nuclear translocation. The expression levels of pro-inflammatory cytokines, such as TNF-α and IL-6, decreased significantly. In conclusion, these results suggest that FGF21 protects the aging mice brain from d-gal-induced injury by attenuating oxidative stress damage and decreasing AGE formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts.

    Directory of Open Access Journals (Sweden)

    Olga N Karpus

    Full Text Available CD55 (decay-accelerating factor is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS. CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS.FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (dsRNA sensors melanoma differentiation-associated gene 5 (MDA5 and retinoic acid-inducible gene-I (RIG-I. CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry.CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA, osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C. Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55.Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

  16. Protection of ionizing radiation-induced cytogenetic damage by hydroalcoholic extract of Cynodon dactylon in Chinese hamster lung fibroblast cells and human peripheral blood lymphocytes.

    Science.gov (United States)

    Rao, Bola Sadashiva Satish; Upadhya, Dinesh; Adiga, Satish Kumar

    2008-01-01

    The radiomodulatory potential of hydroalcoholic extract of a medicinal plant Cynodon dactylon (family: Poaceae) against radiation-induced cytogenetic damage was analyzed using Chinese hamster lung fibroblast (V79) cells and human peripheral blood lymphocytes (HPBLs) growing in vitro. Induction of micronuclei was used as an index of cytogenetic damage, evaluated in cytokinesis blocked binucleate cells. The hydroalcoholic Cynodon dactylon extract (CDE) rendered protection against the radiation-induced DNA damage, as evidenced by the significant (p<0.001) reduction in micronucleated binucleate cells (MNBNC%) after various doses of CDE treatment in V79 cells and HPBLs. The optimum dose of CDE (40 and 50 microg/ml in HPBLs and V79 cells, respectively) with the greatest reduction in micronuclei was further used in combination with various doses of gamma radiation (0.5, 1, 2, 3, and 4 Gy) exposed 1 h after CDE treatment. A linear dose-dependent MNBNC% increase in radiation alone group was observed, while 40/50 microg/ml CDE significantly resulted in the reduction of MNBNC%, compared to the respective radiation alone groups. CDE resulted in a dose-dependent increase in free radical scavenging ability against various free radicals, viz., 2, 2-diphenyl-2-picryl-hydrazyl (DPPH); 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); superoxide anion (O2*-); hydroxyl radical (OH*) and nitric oxide radical (NO*) generated in vitro. Also, an excellent (70%) inhibition of lipid peroxidation in vitro was observed at a dose of 300 microg/ml CDE, attaining the saturation point at higher doses. The present findings demonstrated the radioprotective effect of CDE, also rendering protection against radiation-induced genomic instability and DNA damage. The observed radioprotective effect may be partly attributed to the free radical scavenging and antilipid peroxidative potential of CDE.

  17. Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

    Science.gov (United States)

    Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Whitaker-Menezes, Diana; Daumer, Kristin M; Milliman, Janet N; Chiavarina, Barbara; Migneco, Gemma; Witkiewicz, Agnieszka K; Martinez-Cantarin, Maria P; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Lisanti, Michael P; Sotgia, Federica

    2010-06-15

    Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti

  18. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  19. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    International Nuclear Information System (INIS)

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-01-01

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl 2 -induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical

  20. A correlation between ultraviolet-induced sister chromatid exchanges and ultraviolet-indced mutagenesis in ''Muntiacus muntjak'' (Indian Muntjac) skin fibroblasts in culture

    International Nuclear Information System (INIS)

    Lugo, M.H.

    1983-01-01

    The purpose of this thesis was to develop the capability of simultaneously assaying SCEs and mutations in Indian muntjac cells to determine (1) the relationship between the ultraviolet radiation (UVR) induction of SCEs and the UVR induction of mutations at the (UVR) HGPRT locus in Indian muntjac cells and (2) the possible role of DNA repair in the UVR induction of these two events. Indian muntjac skin fibroblasts were chosen for this study because of a unique karyotype consisting of a diploid chromosome number of 6 in females and 7 in males. An HGPRT mutation assay in Indian muntjac cells was developed by this author since at the time this study was undertaken no mutational assay system utilizing Indian muntjac cells existed. It is concluded from this study that a linear correlation exists betwen the UVR-induction of SCEs and of mutations to 6TG resistance in Indian muntjac cells. As more time is allowed between the UVR-induced DNA damage and onset of DNA replication, more of the lesions leading to both mutations and SCE formation are repaired. The fact that SCE and mutation frequencies are reduced at different rates may indicate that the lesions responsible for SCEs and for mutations are repaired differently

  1. Extracellular matrix metalloproteinase inducer (EMMPRIN) expression correlates positively with active angiogenesis and negatively with basic fibroblast growth factor expression in epithelial ovarian cancer.

    Science.gov (United States)

    Szubert, Sebastian; Szpurek, Dariusz; Moszynski, Rafal; Nowicki, Michal; Frankowski, Andrzej; Sajdak, Stefan; Michalak, Slawomir

    2014-03-01

    The primary aim of this paper was to evaluate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its relationship with proangiogenic factors and microvessel density (MVD) in ovarian cancer. The study group included 58 epithelial ovarian cancers (EOCs), 35 benign ovarian tumors, and 21 normal ovaries. The expression of EMMPRIN, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was assessed by ELISA of tissue homogenates. Antibodies against CD105, CD31, and CD34 were used to immunohistochemically assess MVD. We have found significantly higher EMMPRIN expression in EOC than in benign ovarian tumors and normal ovaries. Similarly, the VEGF expression was higher in EOC than in benign ovarian tumors and normal ovaries. By contrast, bFGF expression was lower in EOC than in benign ovarian tumors and ovary samples. EMMPRIN expression in EOC was directly correlated with VEGF expression and CD105-MVD, but inversely correlated with bFGF expression. Grade 2/3 ovarian cancers had increased expression of EMMPRIN and VEGF, increased CD105-MVD, and lowered expression of bFGF compared to grade 1 ovarian cancers. Moreover, EMMPRIN expression was higher in advanced (FIGO III and IV) ovarian cancer. The upregulation of EMMPRIN and VEGF expression is correlated with increased CD105-MVD and silenced bFGF, which suggests early and/or reactivated angiogenesis in ovarian cancer. Aggressive EOC is characterized by the following: high expression of EMMPRIN and VEGF, high CD105-MVD, and low expression of bFGF.

  2. Identification of titanium dioxide nanoparticles in food products: induce intracellular oxidative stress mediated by TNF and CYP1A genes in human lung fibroblast cells.

    Science.gov (United States)

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Al-Hadi, Ahmed M; Juhaimi, Fahad Al; Mahmoud, Mohamed H; Alshatwi, Ali A

    2015-01-01

    Food grade TiO2 (E171) is a synthetic additive, and widely used as a coloring agent in many foods, pharmaceutical and personal care products. A few reports have highlighted that insoluble particulates (less than 200nm) of food grade TiO2 are found in many foods and confectionary products. However, information regarding the physico-chemical properties (i.e., size and shape)-based food grade TiO2 nanotoxicity related human health issues are limited. The main goal of this study is to examine the presence of nano-sized particulates and its structural characteristics of food grade- TiO2 materials and to assess the acute cellular uptake and metabolic stress induced by these particulates in human lung fibroblast (WI-38) cells. The results of transmission electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction studies indicated that about food grade TiO2 sample contains spherical shaped particulate forms in the nano-scale range, S>G0/G1) and changes in the TNF and CYP1A gene expression pattern are linked to cellular stress. Thus, food grade TiO2 as nano-scaled contaminants could not only be potential human health risk factors, suggesting that safety considerations with special respect to a few crucial factors such as size, and shape should be considered and regulated by food regulators. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

    Science.gov (United States)

    Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

    2009-09-01

    Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

  4. A low-protein diet induces body weight loss and browning of subcutaneous white adipose tissue through enhanced expression of hepatic fibroblast growth factor 21 (FGF21).

    Science.gov (United States)

    Pérez-Martí, Albert; Garcia-Guasch, Maite; Tresserra-Rimbau, Anna; Carrilho-Do-Rosário, Alexandra; Estruch, Ramon; Salas-Salvadó, Jordi; Martínez-González, Miguel Ángel; Lamuela-Raventós, Rosa; Marrero, Pedro F; Haro, Diego; Relat, Joana

    2017-08-01

    Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic candidate for the treatment of obesity. Since FGF21 production is regulated by various nutritional factors, we analyze the impact of low protein intake on circulating levels of this growth hormone in mice and in a sub cohort of the PREDIMED (Prevención con Dieta Mediterránea) trial. We also describe the role of hepatic FGF21 in metabolic adaptation to a low-protein diet (LPD). We fed control and liver-specific Fgf21 knockout (LFgf21KO) mice a LPD. This diet increased FGF21 production by inducing its overexpression in liver, and this correlated with a body weight decrease without changes in food intake. The LPD also caused FGF21-dependent browning in subcutaneous white adipose tissue (scWAT), as indicated by an increase in the expression of uncoupling protein 1 (UCP1). In a subgroup of 78 individuals from the PREDIMED trial, we observed an inverse correlation between protein intake and circulating FGF21 levels. Our results reinforce the involvement of FGF21 in coordinating energy homeostasis under a range of nutritional conditions. Moreover, here we describe an approach to increase the endogenous production of FGF21, which if demonstrated functional in humans, could generate a treatment for obesity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts.

    Science.gov (United States)

    Lybæk, Helle; de Bruijn, Diederik; den Engelsman-van Dijk, Anke H A; Vanichkina, Darya; Nepal, Chirag; Brendehaug, Atle; Houge, Gunnar

    2014-03-01

    It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of "chromatin-CGH") and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14-15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.

  6. TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

    Science.gov (United States)

    Kucab, Jill E; Zwart, Edwin P; van Steeg, Harry; Luijten, Mirjam; Schmeiser, Heinz H; Phillips, David H; Arlt, Volker M

    2016-03-01

    3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

    Science.gov (United States)

    Jia, Xiao-Yi; Chang, Yan; Sun, Xiao-Jing; Wu, Hua-Xun; Wang, Chun; Xu, Hong-Mei; Zhang, Lei; Zhang, Ling-Ling; Zheng, Yong-Qiu; Song, Li-Hua; Wei, Wei

    2014-01-01

    The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1β (10ng/ml) in vitro. TGP (12.5 and 62.5μg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis. © 2013.

  8. Methyl salicylate lactoside inhibits inflammatory response of fibroblast-like synoviocytes and joint destruction in collagen-induced arthritis in mice.

    Science.gov (United States)

    Xin, Wenyu; Huang, Chao; Zhang, Xue; Xin, Sheng; Zhou, Yiming; Ma, Xiaowei; Zhang, Dan; Li, Yongjie; Zhou, Sibai; Zhang, Dongming; Zhang, Tiantai; Du, Guanhua

    2014-07-01

    Methyl salicylate 2-O-β-d-lactoside (MSL), whose chemical structure is similar to that of salicylic acid, is a natural product derivative isolated from a traditional Chinese herb. The aim of this study was to investigate the therapeutic effect of MSL in mice with collagen-induced arthritis (CIA) and explore its underlying mechanism. The anti-arthritic effects of MSL were evaluated on human rheumatoid fibroblast-like synoviocytes (FLS) in vitro and CIA in mice in vivo by obtaining clinical scores, measuring hind paw thickness and inflammatory cytokine levels, radiographic evaluations and histopathological assessments. Treatment with MSL after the onset of arthritis significantly prevented the progression and development of rheumatoid arthritis (RA) in CIA mice without megascopic gastric mucosa damage. In addition, MSL inhibited the production of pro-inflammatory mediators, the phosphorylation and translocation of NF-κB, and cell proliferation induced by TNF-α in FLS. MSL non-selectively inhibited the activity of COX in vitro, but was a more potent inhibitor of COX-2 than COX-1. MSL also inhibited the phosphorylation of inhibitor of NF-κB kinase, IκBα and p65, thus blocking the nuclear translocation of NF-κB in TNF-α-stimulated FLS. MSL exerts therapeutic effects on CIA mice, suppressing the inflammatory response and joint destruction by non-selectively inhibiting the activity of COX and suppressing activation of the NF-κB signalling pathway, but without damaging the gastric mucosa. Therefore, MSL has great potential to be developed into a novel therapeutic agent for the treatment of RA. © 2014 The British Pharmacological Society.

  9. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

  10. Persistent tumor-induced osteomalacia confirmed by elevated postoperative levels of serum fibroblast growth factor-23 and 5-year follow-up of bone density changes.

    Science.gov (United States)

    Zimering, Mark B; Caldarella, Felice A; White, Kenneth E; Econs, Michael J

    2005-01-01

    To describe a case of persistent tumor-induced osteomalacia, determine whether serum fibroblast growth factor-23 (FGF-23) levels postoperatively indicate incomplete tumor resection, and report lumbar spine and forearm bone mineral density (BMD) changes during 5 years of follow-up. We present clinical, radiologic, histologic, and bone densitometry data as well as serum FGF-23 levels (determined with use of a novel C-terminal enzyme-linked immunosorbent assay) from the study patient and discuss these findings in the context of previous literature. A 52-year-old man, who presented with muscle weakness and multiple fractures, was found to have low values for serum phosphorus, serum 1,25-dihydroxyvitamin D, and maximal tubular reabsorption of phosphate per glomerular filtration rate, a high level of serum alkaline phosphatase, and a normal serum concentration of parathyroid hormone, characteristic of tumor-induced osteomalacia. Magnetic resonance imaging to evaluate an abnormality of the left foot revealed a soft tissue mass, biopsy of which confirmed the presence of a benign, phosphaturic, mesenchymal tumor. The baseline serum FGF-23 level (2,050 RU/mL) was more than 17 times the upper limit of normal for adults (23 to 118 RU/mL) and decreased substantially within 1 day after partial resection of the tumor but remained above normal postoperatively. BMD changes indicated rapid substantial recovery of vertebral BMD but ongoing loss of forearm bone density. The serum FGF-23 level is high in a substantial proportion of patients with tumor-induced osteomalacia. The postoperative above normal levels of serum FGF-23 correlated with known persistence of tumor in our study patient. In a patient with normal renal function, such as our study patient, levels of serum FGF-23 studied with use of the C-terminal enzyme-linked immunosorbent assay reached their nadir within 24 hours postoperatively. This result suggests that this assay can provide clinicians with rapid prognostic

  11. The key role of miR-21-regulated SOD2 in the medium-mediated bystander responses in human fibroblasts induced by α-irradiated keratinocytes

    International Nuclear Information System (INIS)

    Tian, Wenqian; Yin, Xiaoming; Wang, Longxiao; Wang, Jingdong; Zhu, Wei; Cao, Jianping; Yang, Hongying

    2015-01-01

    Highlights: • After co-culture with α-irradiated HaCaT cells, WS1 cells displayed oxidative stress and DNA damage. • Increased miR-21 expression in bystander cells was critical to the occurrence of RIBEs. • SOD2 of bystander cells played an important role in bystander responses. • miR-21 mediated bystander effects through its regulation on SOD2. - Abstract: Radiation-induced bystander effect (RIBE) is well accepted in the radiation research field by now, but the underlying molecular mechanisms for better understanding this phenomenon caused by intercellular communication and intracellular signal transduction are still incomplete. Although our previous study has demonstrated an important role of miR-21 of unirradiated bystander cells in RIBEs, the direct evidence for the hypothesis that RIBE is epigenetically regulated is still limited and how miR-21 mediates RIBEs is unknown. Reactive oxygen species (ROS) have been demonstrated to be involved in RIBEs, however, the roles of anti-oxidative stress system of cells in RIBEs are unclear. Using transwell insert co-culture system, we investigated medium-mediated bystander responses in WS1 human fibroblasts after co-culture with HaCaT keratinocytes traversed by α-particles. Results showed that the ROS levels in unirradiated bystander WS1 cells were significantly elevated after 30 min of co-culture, and 53BP1 foci, a surrogate marker of DNA damage, were obviously induced after 3 h of co-culture. This indicates the occurrence of oxidative stress and DNA damage in bystander WS1 cells after co-culture with irradiated keratinocytes. Furthermore, the expression of miR-21 was increased in bystander WS1 cells, downregulation of miR-21 eliminated the bystander responses, overexpression of miR-21 alone could induce bystander-like oxidative stress and DNA damage in WS1 cells. These data indicate an important mediating role of miR-21 in RIBEs. In addition, MnSOD or SOD2 in WS1 cells was involved in the bystander effects

  12. The key role of miR-21-regulated SOD2 in the medium-mediated bystander responses in human fibroblasts induced by α-irradiated keratinocytes.

    Science.gov (United States)

    Tian, Wenqian; Yin, Xiaoming; Wang, Longxiao; Wang, Jingdong; Zhu, Wei; Cao, Jianping; Yang, Hongying

    2015-10-01

    Radiation-induced bystander effect (RIBE) is well accepted in the radiation research field by now, but the underlying molecular mechanisms for better understanding this phenomenon caused by intercellular communication and intracellular signal transduction are still incomplete. Although our previous study has demonstrated an important role of miR-21 of unirradiated bystander cells in RIBEs, the direct evidence for the hypothesis that RIBE is epigenetically regulated is still limited and how miR-21 mediates RIBEs is unknown. Reactive oxygen species (ROS) have been demonstrated to be involved in RIBEs, however, the roles of anti-oxidative stress system of cells in RIBEs are unclear. Using transwell insert co-culture system, we investigated medium-mediated bystander responses in WS1 human fibroblasts after co-culture with HaCaT keratinocytes traversed by α-particles. Results showed that the ROS levels in unirradiated bystander WS1 cells were significantly elevated after 30min of co-culture, and 53BP1 foci, a surrogate marker of DNA damage, were obviously induced after 3h of co-culture. This indicates the occurrence of oxidative stress and DNA damage in bystander WS1 cells after co-culture with irradiated keratinocytes. Furthermore, the expression of miR-21 was increased in bystander WS1 cells, downregulation of miR-21 eliminated the bystander responses, overexpression of miR-21 alone could induce bystander-like oxidative stress and DNA damage in WS1 cells. These data indicate an important mediating role of miR-21 in RIBEs. In addition, MnSOD or SOD2 in WS1 cells was involved in the bystander effects, overexpression of SOD2 abolished the bystander oxidative stress and DNA damage, indicating that SOD2 was critical to the induction of RIBEs. Moreover, we found that miR-21 regulated SOD2, suggesting that miR-21 might mediate bystander responses through its regulation on SOD2. In conclusion, this study revealed a profound role of miR-21-regulated SOD2 of unirradiated WS1

  13. The key role of miR-21-regulated SOD2 in the medium-mediated bystander responses in human fibroblasts induced by α-irradiated keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Wenqian; Yin, Xiaoming; Wang, Longxiao; Wang, Jingdong; Zhu, Wei; Cao, Jianping [School of Radiation Medicine and Protection, Medical College of Soochow University/Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, 199 Renai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province 215123 (China); Yang, Hongying, E-mail: yanghongying@suda.edu.cn [School of Radiation Medicine and Protection, Medical College of Soochow University/Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, 199 Renai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province 215123 (China); Institute of Radiotherapy & Oncology, Soochow University (China)

    2015-10-15

    Highlights: • After co-culture with α-irradiated HaCaT cells, WS1 cells displayed oxidative stress and DNA damage. • Increased miR-21 expression in bystander cells was critical to the occurrence of RIBEs. • SOD2 of bystander cells played an important role in bystander responses. • miR-21 mediated bystander effects through its regulation on SOD2. - Abstract: Radiation-induced bystander effect (RIBE) is well accepted in the radiation research field by now, but the underlying molecular mechanisms for better understanding this phenomenon caused by intercellular communication and intracellular signal transduction are still incomplete. Although our previous study has demonstrated an important role of miR-21 of unirradiated bystander cells in RIBEs, the direct evidence for the hypothesis that RIBE is epigenetically regulated is still limited and how miR-21 mediates RIBEs is unknown. Reactive oxygen species (ROS) have been demonstrated to be involved in RIBEs, however, the roles of anti-oxidative stress system of cells in RIBEs are unclear. Using transwell insert co-culture system, we investigated medium-mediated bystander responses in WS1 human fibroblasts after co-culture with HaCaT keratinocytes traversed by α-particles. Results showed that the ROS levels in unirradiated bystander WS1 cells were significantly elevated after 30 min of co-culture, and 53BP1 foci, a surrogate marker of DNA damage, were obviously induced after 3 h of co-culture. This indicates the occurrence of oxidative stress and DNA damage in bystander WS1 cells after co-culture with irradiated keratinocytes. Furthermore, the expression of miR-21 was increased in bystander WS1 cells, downregulation of miR-21 eliminated the bystander responses, overexpression of miR-21 alone could induce bystander-like oxidative stress and DNA damage in WS1 cells. These data indicate an important mediating role of miR-21 in RIBEs. In addition, MnSOD or SOD2 in WS1 cells was involved in the bystander effects

  14. N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

    LENUS (Irish Health Repository)

    Burke, John P

    2012-02-01

    BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

  15. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  16. Patient-Derived Human Induced Pluripotent Stem Cells From Gingival Fibroblasts Composited With Defined Nanohydroxyapatite/Chitosan/Gelatin Porous Scaffolds as Potential Bone Graft Substitutes.

    Science.gov (United States)

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Zhang, Junfeng; Qin, Haiyan; Hu, Qingang

    2016-01-01

    Human embryonic stem cells and adult stem cells have always been the cell source for bone tissue engineering. However, their limitations are obvious, including ethical concerns and/or a short lifespan. The use of human induced pluripotent stem cells (hiPSCs) could avoid these problems. Nanohydroxyapatite (nHA) is an important component of natural bone and bone tissue engineering scaffolds. However, its regulation on osteogenic differentiation with hiPSCs from human gingival fibroblasts (hGFs) is unknown. The purpose of the present study was to investigate the osteogenic differentiation of hiPSCs from patient-derived hGFs regulated by nHA/chitosan/gelatin (HCG) scaffolds with different nHA ratios, such as HCG-111 (1 wt/vol% nHA) and HCG-311 (3 wt/vol% nHA). First, hGFs were reprogrammed into hiPSCs, which have enhanced osteogenic differentiation capability. Second, HCG-111 and HCG-311 scaffolds were successfully synthesized. Finally, hiPSC/HCG complexes were cultured in vitro or subcutaneously transplanted into immunocompromised mice in vivo. The osteogenic differentiation effects of two types of HCG scaffolds on hiPSCs were assessed for up to 12 weeks. The results showed that HCG-311 increased osteogenic-related gene expression of hiPSCs in vitro proved by quantitative real-time polymerase chain reaction, and hiPSC/HCG-311 complexes formed much bone-like tissue in vivo, indicated by cone-beam computed tomography imaging, H&E staining, Masson staining, and RUNX-2, OCN immunohistochemistry staining. In conclusion, our study has shown that osteogenic differentiation of hiPSCs from hGFs was improved by HCG-311. The mechanism might be that the nHA addition stimulates osteogenic marker expression of hiPSCs from hGFs. Our work has provided an innovative autologous cell-based bone tissue engineering approach with soft tissues such as clinically abundant gingiva. The present study focused on patient-personalized bone tissue engineering. Human induced pluripotent stem cells

  17. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

    Science.gov (United States)

    Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2014-02-23

    Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the

  18. Supernatants from oral epithelial cells and gingival fibroblasts modulate human immunodeficiency virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages.

    Science.gov (United States)

    González, O A; Ebersole, J L; Huang, C B

    2010-04-01

    Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes

  19. Transcription Factor SOX5 Promotes the Migration and Invasion of Fibroblast-Like Synoviocytes in Part by Regulating MMP-9 Expression in Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Yumeng Shi

    2018-04-01

    Full Text Available ObjectivesFibroblast-like synoviocytes (FLS exhibit a unique aggressive phenotype in rheumatoid arthritis (RA. Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated.MethodsThe migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9, chemokines (CCL4, CCL2, CCR5 and CCR2, and pro-inflammatory cytokines (TNF-α and IL-6, were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA in DBA/1J mice.ResultsKnockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice.ConclusionSOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.

  20. Transcription Factor SOX5 Promotes the Migration and Invasion of Fibroblast-Like Synoviocytes in Part by Regulating MMP-9 Expression in Collagen-Induced Arthritis.

    Science.gov (United States)

    Shi, Yumeng; Wu, Qin; Xuan, Wenhua; Feng, Xiaoke; Wang, Fang; Tsao, Betty P; Zhang, Miaojia; Tan, Wenfeng

    2018-01-01

    Fibroblast-like synoviocytes (FLS) exhibit a unique aggressive phenotype in rheumatoid arthritis (RA). Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated. The migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9), chemokines (CCL4, CCL2, CCR5 and CCR2), and pro-inflammatory cytokines (TNF-α and IL-6), were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP) studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA) in DBA/1J mice. Knockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice. SOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.

  1. Tumor-induced osteomalacia with elevated fibroblast growth factor 23: a case of phosphaturic mesenchymal tumor mixed with connective tissue variants and review of the literature.

    Science.gov (United States)

    Hu, Fang-Ke; Yuan, Fang; Jiang, Cheng-Ying; Lv, Da-Wei; Mao, Bei-Bei; Zhang, Qiang; Yuan, Zeng-Qiang; Wang, Yan

    2011-11-01

    Tumor-induced osteomalacia (TIO), or oncogenic osteomalacia (OOM), is a rare acquired paraneoplastic disease characterized by renal phosphate wasting and hypophosphatemia. Recent evidence shows that tumor-overexpressed fibroblast growth factor 23 (FGF23) is responsible for the hypophosphatemia and osteomalacia. The tumors associated with TIO are usually phosphaturic mesenchymal tumor mixed connective tissue variants (PMTMCT). Surgical removal of the responsible tumors is clinically essential for the treatment of TIO. However, identifying the responsible tumors is often difficult. Here, we report a case of a TIO patient with elevated serum FGF23 levels suffering from bone pain and hypophosphatemia for more than three years. A tumor was finally located in first metacarpal bone by octreotide scintigraphy and she was cured by surgery. After complete excision of the tumor, serum FGF23 levels rapidly decreased, dropping to 54.7% of the preoperative level one hour after surgery and eventually to a little below normal. The patient's serum phosphate level rapidly improved and returned to normal level in four days. Accordingly, her clinical symptoms were greatly improved within one month after surgery. There was no sign of tumor recurrence during an 18-month period of follow-up. According to pathology, the tumor was originally diagnosed as "lomangioma" based upon a biopsy sample, "proliferative giant cell tumor of tendon sheath" based upon sections of tumor, and finally diagnosed as PMTMCT by consultation one year after surgery. In conclusion, although an extremely rare disease, clinicians and pathologists should be aware of the existence of TIO and PMTMCT, respectively.

  2. Benzo(a)pyrene metabolism, DNA-binding and UV-induced repair of DNA damage in cultured skin fibroblasts from a patient with unilateral multiple basal cell carcinoma

    International Nuclear Information System (INIS)

    Don, P.S.C.; Mukhtar, H.; Das, M.; Berger, N.A.; Bickers, D.R.

    1989-01-01

    The metabolism of benzo(a)pyrene (BP), and its subsequent binding to DNA, and the repair of UV-induced DNA damage were studied in fibroblasts cultured from the skin of a 61-year-old male who had multiple basal cell carcinoma (BCC) (>100) on his left upper trunk. Results suggest that BP metabolism and repair of DNA are altered in tumor-bearing site (TSB) cells and that patients with this type of metabolic profile may be at higher risk of the development of cutaneous neoplasms. It is also possible that fibroblasts from tumour bearing skin undergo some as yet unexplained alteration in carcinogen metabolism as a consequence of the induction of neoplasia. (author)

  3. Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts

    International Nuclear Information System (INIS)

    Antoniades, H.N.; Galanopoulos, T.; Neville-Golden, J.; Kiritsy, C.P.; Lynch, S.E.

    1991-01-01

    Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study, the authors have demonstrated with in situ hydridization and immunocytochemistry the reversible expression of 3-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. These studies provide a mulecular basis for understanding the mechanisms contributing to normal tissue repair. They suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that chronic injury may induce irreversible gene expression leading to pathologic, unregulated cell growth

  4. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  5. Sirt1 Protects against Oxidative Stress-Induced Apoptosis in Fibroblasts from Psoriatic Patients: A New Insight into the Pathogenetic Mechanisms of Psoriasis

    Directory of Open Access Journals (Sweden)

    Matteo Becatti

    2018-05-01

    Full Text Available Psoriasis, a multisystem chronic disease characterized by abnormal keratinocyte proliferation, has an unclear pathogenesis where systemic inflammation and oxidative stress play mutual roles. Dermal fibroblasts, which are known to provide a crucial microenvironment for epidermal keratinocyte function, represented the selected experimental model in our study which aimed to clarify the potential role of SIRT1 in the pathogenetic mechanisms of the disease. We firstly detected the presence of oxidative stress (lipid peroxidation and total antioxidant capacity, significantly reduced SIRT1 expression level and activity, mitochondrial damage and apoptosis (caspase-3, -8 and -9 activities in psoriatic fibroblasts. Upon SIRT1 activation, redox balance was re-established, mitochondrial function was restored and apoptosis was no longer evident. Furthermore, we examined p38, ERK and JNK activation, which was strongly altered in psoriatic fibroblasts, in response to SIRT1 activation and we measured caspase-3 activity in the presence of specific MAPK inhibitors demonstrating the key role of the SIRT1 pathway against apoptotic cell death via MAPK modulation. Our results clearly demonstrate the involvement of SIRT1 in the protective mechanisms related to fibroblast injury in psoriasis. SIRT1 activation exerts an active role in restoring both mitochondrial function and redox balance via modulation of MAPK signaling. Hence, SIRT1 can be proposed as a specific tool for the treatment of psoriasis.

  6. Epidermal growth factor receptor-induced activato protein 1 activity controls density-dependent growht inhibition in normal rat kidney fibroblasts.

    NARCIS (Netherlands)

    Hornberg, J.J.; Dekker, H.; Peters, P.H.J.; Langerak, P.; Westerhoff, H.V.; Lankelma, J.; Zoelen, E.J.J.

    2006-01-01

    Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these

  7. Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling

    NARCIS (Netherlands)

    Kern, Georg; Mair, Sabine M; Noppert, Susie-Jane; Jennings, Paul; Schramek, Herbert; Rudnicki, Michael; Mueller, Gerhard A; Mayer, Gert; Koppelstaetter, Christian

    2014-01-01

    Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the

  8. Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts

    International Nuclear Information System (INIS)

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Qin, Haiyan; Hu, Qingang; Zhang, Junfeng; Dong, Lei

    2015-01-01

    Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson’s staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson’s, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

  9. Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts.

    Science.gov (United States)

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Zhang, Junfeng; Dong, Lei; Qin, Haiyan; Hu, Qingang

    2015-07-08

    Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson's staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson's, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

  10. Parapoxvirus orf virus infection induces an increase in interleukin-8, tumour necrosis factor-α, and decorin in goat skin fibroblast cells

    Directory of Open Access Journals (Sweden)

    Wang Lingling

    2016-09-01

    Full Text Available Introduction: Orf virus (ORFV is a prototype Parapoxvirus species in the Poxviridae family that causes serious zoonotic infectious disease. Goat skin fibroblast (GSF cells are the major host targets of ORFV. Interleukin 8 (IL-8 and tumour necrosis factor (TNF-α are known to play a vital role in immune response during viral infections. However, the manner of variation over time of their level of expression in GSF cells remains unclear.

  11. In vitro studies to evaluate the antioxidant property of salidroside and rosavin and protective effects of electron beam radiation induced damages in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Tejashvi, Kedilaya R.; Padma, Shetty K.; Suchetha Kumari, N.

    2014-01-01

    Rosavin and Salidroside are active component of Rhodiola rosea, it is a phenylpropanoid derivative of plant. Rhodiola rosea, also known as 'golden root' or 'roseroot' belongs to the plant family Crassulaceae. Rhodiola grows primarily in dry sandy ground at high altitudes in the arctic areas of Europe and Asia. Plant is rich with phenolic compounds, known to have a strong antioxidant property. Studies have shown that Rhodiola rosea has a capacity to decrease toxicity of Adriamycin (anti-cancer drugs), while it enhances their anti-carcinogenic effects. Enhanced antioxidant activity of Rhodiola rosea play role in the prevention of both chronic disease and aging. Present study is aimed to determine the antioxidant property of Rosavin and Salidroside and dose determination on human dermal fibroblast against dermal fibroblast. Rosavin and Salidroside were dissolved in 10% DMSO. Invitro biochemical assays like DPPH radical scavenging assay, Ferric Anion Reducing Potential using TPTZ, Nitric Oxide scavenging assay, Total antioxidant determination assay, Super Anion Radical Scavenging assays were carried out to know property of the extract. Extracts were then treated on monolayer dermal fibroblast cells survival assay was performed. Salidroside has shown 80% total antioxidant property compare to Rosavin with respect Ascorbic acid as a standard. 100'R concentration of Salidroside and Rosavin has quite equal potential to scavenging DPPH similar like Ascorbic acid. Ferric Anion Reducing Potential using TPTZ, Nitric Oxide scavenging assays have also shown both Salidroside and Rosavin has a good antioxidant property. Invitro studies on dermal fibroblast have shown remarkable protective effect on normal and irradiated groups. (author)

  12. Bioactive reagents used in mesotherapy for skin rejuvenation in vivo induce diverse physiological processes in human skin fibroblasts in vitro- a pilot study.

    Science.gov (United States)

    Jäger, Claudia; Brenner, Christiane; Habicht, Jüri; Wallich, Reinhard

    2012-01-01

    The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135(®) and NCTF135HA(®) , cell proliferation was comparable to control cultures; however, with higher expression of collagen type-1, matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, addition of Soluvit(®) N and Meso-BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro. © 2011 John Wiley & Sons A/S.

  13. ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

    International Nuclear Information System (INIS)

    Park, Jun; Jo, Yong Hwa; Cho, Chang Hoon; Choe, Wonchae; Kang, Insug; Baik, Hyung Hwan; Yoon, Kyung-Sik

    2013-01-01

    Highlights: ► A-T cells were not hypersensitive to low levels of DNA DSBs. ► A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. ► A-T cells underwent premature senescence after DNA damage accumulated. ► Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-β-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.

  14. Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblasts.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Holmsen, H

    1990-09-01

    Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.

  15. Fibroblast growth factor 23

    African Journals Online (AJOL)

    Dr Olaleye

    Systemic phosphate homeostasis is maintained through several hormonal mechanisms which involve fibroblast growth factor 23 (FGF-23), α-klotho, vitamin D and parathyroid hormone. FGF-23 is known to be the major regulator of phosphate balance (Mirams et al., 2004). FGF-23 is a phosphaturic hormone, which is.

  16. Rac inhibition reverses the phenotype of fibrotic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shi-wen Xu

    Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

  17. Shell extracts of the edible mussel and oyster induce an enhancement of the catabolic pathway of human skin fibroblasts, in vitro.

    Science.gov (United States)

    Latire, Thomas; Legendre, Florence; Bouyoucef, Mouloud; Marin, Frédéric; Carreiras, Franck; Rigot-Jolivet, Muriel; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

    2017-10-01

    Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity.

  18. Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Jong Yuh Cherng

    2012-08-01

    Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm2. After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

  19. LXA4 actions direct fibroblast function and wound closure

    International Nuclear Information System (INIS)

    Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

    2015-01-01

    Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

  20. Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

  1. A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

    Science.gov (United States)

    Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

    2013-01-01

    We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

  2. Diagnóstico rápido de citomegalovirus (CMV en pacientes inmunocomprometidos mediante anticuerpos monoclonales que reconocen proteinas precoces virales Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by using monoclonal antibodies against early viral antigens

    Directory of Open Access Journals (Sweden)

    Maritza Alvarez

    1989-06-01

    Full Text Available Se aplicó la técnica de detección de antigenos precoces fluorescentes (DAPF usando el anticuerpo monoclonal E-13 McAb, mediante el cual se lograron detectar 15 casos positivos a CMV de 75 muestras de orina o sangre ("buffy coat" tomadas de 52 pacientes inmunocomprometidos ingresados en el Instituto de Nefrología de ciudad Habana. Aplicando las técnicas clásicas de aislamiento en fibroblastos humanos diploides (MRC-5, se lograron aislar 12 cepas de CMV de casos previamente positivos por DAPF; lográndose además un aislamiento en una muestra reportada negativa por fluorescencia. Se observó una coincidencia de un 80% entre ambas técnicas. Se detectó la presencia de anticuerpos IgG contra CMV en todos los casos estudiados, utilizando para ello la técnica ELISA.A technique was applied to detect early fluorescent antigens (DEFA of cytomegalovirus (CMV using the E13 monoclonal antibodies in 52 immunocompromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat samples taken, 15 were found positive to CMV. Using classical diploide human fibroblast isolation technique, 12 CMV strains were isloation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.

  3. Label-free characterization of ultra violet-radiation-induced changes in skin fibroblasts with Raman spectroscopy and quantitative phase microscopy.

    Science.gov (United States)

    Singh, S P; Kang, Sungsam; Kang, Jeon Woong; So, Peter T C; Dasari, Ramanchandra Rao; Yaqoob, Zahid; Barman, Ishan

    2017-09-07

    Minimizing morbidities and mortalities associated with skin cancers requires sustained research with the goal of obtaining fresh insights into disease onset and progression under specific stimuli, particularly the influence of ultraviolet rays. In the present study, label-free profiling of skin fibroblasts exposed to time-bound ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Statistically significant differences in quantifiable biophysical parameters, such as matter density and cell dry mass, were observed with phase imaging. Accurate estimation of changes in the biochemical constituents, notably nucleic acids and proteins, was demonstrated through a combination of Raman spectroscopy and multivariate analysis of spectral patterns. Overall, the findings of this study demonstrate the promise of these non-perturbative optical modalities in accurately identifying cellular phenotypes and responses to external stimuli by combining molecular and biophysical information.

  4. The NF-κB family member RelB regulates microRNA miR-146a to suppress cigarette smoke-induced COX-2 protein expression in lung fibroblasts.

    Science.gov (United States)

    Zago, Michela; Rico de Souza, Angela; Hecht, Emelia; Rousseau, Simon; Hamid, Qutayba; Eidelman, David H; Baglole, Carolyn J

    2014-04-21

    Diseases due to cigarette smoke exposure, including chronic obstructive pulmonary disease (COPD) and lung cancer, are associated with chronic inflammation typified by the increased expression of cyclooxygenase-2 (COX-2) protein. RelB is an NF-κB family member that suppresses cigarette smoke induction of COX-2 through an unknown mechanism. The ability of RelB to regulate COX-2 expression may be via miR-146a, a miRNA that attenuates COX-2 in lung fibroblasts. In this study we tested whether RelB attenuation of cigarette smoke-induced COX-2 protein is due to miR-146a. Utilizing pulmonary fibroblasts deficient in RelB expression, together with siRNA knock-down of RelB, we show the essential role of RelB in diminishing smoke-induced COX-2 protein expression despite robust activation of the canonical NF-κB pathway and subsequent induction of Cox-2 mRNA. RelB did not regulate COX-2 protein expression at the level of mRNA stability. Basal levels of miR-146a were significantly lower in Relb-deficient cells and cigarette smoke increased miR-146a expression only in Relb-expressing cells. Inhibition of miR-146a had no effects on Relb expression or induction of Cox-2 mRNA by cigarette smoke but significantly increased COX-2 protein. These data highlight the potential of a RelB-miR-146a axis as a novel regulatory pathway that attenuates inflammation in response to respiratory toxicants. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  6. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  7. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    International Nuclear Information System (INIS)

    Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto

    2012-01-01

    Highlights: ► ABA is an endogenous hormone in humans, regulating different cell responses. ► ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. ► UV-B irradiation increases ABA content in SSc cultures. ► SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  8. Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts

    International Nuclear Information System (INIS)

    Snyder, R.D.

    1985-01-01

    In an attempt to better understand the mechanism of repair inhibiti