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Sample records for fibroblasts lacking rb1

  1. Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

    Science.gov (United States)

    Hou, Jingang; Kim, Sunchang

    2018-05-05

    Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts.

    Directory of Open Access Journals (Sweden)

    C Herbert Pratt

    2011-03-01

    Full Text Available Lamin A (LMNA is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350 and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670. Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1 activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood.We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (Lmna(Dhe. We found that dermal fibroblasts from heterozygous Lmna(Dhe (Lmna(Dhe/+ mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, Lmna(Dhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3, a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1 also was perturbed in Lmna(Dhe/+ cells. Lmna(Dhe/+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects.These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.

  3. Mitotic Defects Lead to Pervasive Aneuploidy and Accompany Loss of RB1 Activity in Mouse LmnaDhe Dermal Fibroblasts

    Science.gov (United States)

    Pratt, C. Herbert; Curtain, Michelle; Donahue, Leah Rae; Shopland, Lindsay S.

    2011-01-01

    Background Lamin A (LMNA) is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350) and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670). Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1) activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. Results We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (LmnaDhe). We found that dermal fibroblasts from heterozygous LmnaDhe (LmnaDhe/+) mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, LmnaDhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3), a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1) also was perturbed in LmnaDhe /+ cells. LmnaDhe /+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. Conclusions These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control. PMID:21464947

  4. Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus

    2002-01-01

    A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity...

  5. The origin of the RB1 imprint.

    Directory of Open Access Journals (Sweden)

    Deniz Kanber

    Full Text Available The human RB1 gene is imprinted due to a differentially methylated CpG island in intron 2. This CpG island is part of PPP1R26P1, a truncated retrocopy of PPP1R26, and serves as a promoter for an alternative RB1 transcript. We show here by in silico analyses that the parental PPP1R26 gene is present in the analysed members of Haplorrhini, which comprise Catarrhini (Old World Monkeys, Small apes, Great Apes and Human, Platyrrhini (New World Monkeys and tarsier, and Strepsirrhini (galago. Interestingly, we detected the retrocopy, PPP1R26P1, in all Anthropoidea (Catarrhini and Platyrrhini that we studied but not in tarsier or galago. Additional retrocopies are present in human and chimpanzee on chromosome 22, but their distinct composition indicates that they are the result of independent retrotransposition events. Chimpanzee and marmoset have further retrocopies on chromosome 8 and chromosome 4, respectively. To examine the origin of the RB1 imprint, we compared the methylation patterns of the parental PPP1R26 gene and its retrocopies in different primates (human, chimpanzee, orangutan, rhesus macaque, marmoset and galago. Methylation analysis by deep bisulfite sequencing showed that PPP1R26 is methylated whereas the retrocopy in RB1 intron 2 is differentially methylated in all primates studied. All other retrocopies are fully methylated, except for the additional retrocopy on marmoset chromosome 4, which is also differentially methylated. Using an informative SNP for the methylation analysis in marmoset, we could show that the differential methylation pattern of the retrocopy on chromosome 4 is allele-specific. We conclude that the epigenetic fate of a PPP1R26 retrocopy after integration depends on the DNA sequence and selective forces at the integration site.

  6. RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer.

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    Tokuhiro Chano

    2010-06-01

    Full Text Available RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200 plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

  7. Microbial conversion of major ginsenoside Rb1 to minor ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... minor ginsenoside Rd by Indian fermented food bacteria. Kalaiselvi ... South Korea. Accepted 17 April, 2009. Ginsenoside Rb1 is the predominant secondary metabolite (saponin) in Panax ginseng. Hydrolysis of the sugar .... culture and 200 µl of major ginsenoside Rb1. The reaction mixture was incubated ...

  8. Analysis list: RB1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available RB1 Prostate,Uterus + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/R...B1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/RB1.5.tsv http://dbarchive.biosciencedbc.jp/...kyushu-u/hg19/target/RB1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/RB1.Prostate.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/RB1.Uterus.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Prostate.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Uterus.gml ...

  9. Rb1 loss modifies but does not initiate alveolar rhabdomyosarcoma

    Science.gov (United States)

    2013-01-01

    Background Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. Methods We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. Results Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. Conclusions Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function. PMID:24274149

  10. Rapid detection of RB1 recurrent mutations in retinoblastoma by ...

    Indian Academy of Sciences (India)

    In about half of the patients, one mutation is inherited via the germinal cells, while in the .... mutational hot spots in the RB1 gene, making genetic testing complex and challenging ... by direct sequencing. High normal background in sequenc-.

  11. Ginsenoside Rb1 Reduces Nitric Oxide Production via Inhibition of ...

    African Journals Online (AJOL)

    Inhibition of Nuclear Factor-κB Activation in Interleukin-1β- ... 20, 40, 80 µM ginsenoside Rb1. NO concentration was assessed by the Griess reaction. ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, ...

  12. Lack of Day/Night variation in fibroblast growth factor 21 levels in young healthy men.

    Science.gov (United States)

    Foo, J-P; Aronis, K N; Chamberland, J P; Mantzoros, C S

    2015-06-01

    Fibroblast growth factor (FGF) 21 is an endocrine factor with an emerging role as a metabolic regulator. We previously reported the presence of a significant day/night variation of FGF-21 in energy-replete, healthy female subjects. However the day/night patterns of secretion in male subjects remain to be fully elucidated. To elucidate day/night pattern of FGF-21 levels in male subjects in the energy-replete state, its relationship to FFA and to investigate whether a sexual dimorphism exists in FGF-21 physiology. Eight healthy lean male subjects were studied for up to 5 days while on an isocaloric diet. Blood samples were obtained for measurement of FGF-21 and free fatty acids (FFA) hourly from 0800 AM on day 4 till 0800AM on day 5. FGF-21 did not exhibit any statistically significant day/night variation pattern of circulating FGF-21 levels during the isocaloric fed state in male subjects. FGF-21 levels in male subjects are closely cross-correlated with FFA levels, similar to female subjects. A sexual dimorphism exists in FGF-21 physiology; that as opposed to female subjects, no significant day/night variation exists in FGF-21 rhythm in male subjects in the energy-replete state. Circulating pattern of FGF-21, similar to the female subjects, was highly cross-correlated to the FFA levels in the male subjects, signifying that the sexual dimorphism in FGF-21 physiology may be related to the differing lipid metabolism in both the genders.

  13. RB1 mutations and second primary malignancies after hereditary retinoblastoma

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Marees, Tamara; van der Hout, Annemarie H.; Imhof, Saskia M.; Meijers-Heijboer, Hanne; Ringens, Peter J.; van Leeuwen, Flora E.; Moll, Annette C.

    2012-01-01

    Survivors of hereditary retinoblastoma have a high risk of second primary malignancies, but it has not been investigated whether specific RB1 germline mutations are associated with greater risk of second primary malignancies in a large cohort. We conducted a retrospective cohort study of 199

  14. RB1 mutations and second primary malignancies after hereditary retinoblastoma

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Marees, Tamara; van der Hout, Annemarie H.; Imhof, Saskia M.; Meijers-Heijboer, Hanne; Ringens, Peter J.; van Leeuwen, Flora E.; Moll, Annette C.

    Survivors of hereditary retinoblastoma have a high risk of second primary malignancies, but it has not been investigated whether specific RB1 germline mutations are associated with greater risk of second primary malignancies in a large cohort. We conducted a retrospective cohort study of 199

  15. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    in this family revealed skipping of exon 22 in three members of this family. In one proband, a ... This study reveals novel effects of RB1 mutations on splicing and suggests the utility of RNA analysis as an ... of life) and presence of multiple tumors (multifocal). The ..... spliced RNA have been linked to parent of origin as well as.

  16. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    International Nuclear Information System (INIS)

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J.

    2006-01-01

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1α associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome

  17. RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients

    NARCIS (Netherlands)

    Dommering, Charlotte J.; Mol, Berber M.; Moll, Annette C.; Burton, Margaret; Cloos, Jacqueline; Dorsman, Josephine C.; Meijers-Heijboer, Hanne; van der Hout, Annemarie H.

    Background Retinoblastoma (Rb) is a childhood cancer of the retina, commonly initiated by biallelic inactivation of the RB1 gene. Knowledge of the presence of a heritable RB1 mutation can help in risk management and reproductive decision making. We report here on RB1 mutation scanning in a unique

  18. Effects of Ginsenoside Rb1 on Skin Changes

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Kimura

    2012-01-01

    Full Text Available Ginseng roots (Panax ginseng CA Meyer have been used traditionally for the treatment, especially prevention, of various diseases in China, Korea, and Japan. Both experimental and clinical studies suggest ginseng roots to have pharmacological effects in patients with life-style-related diseases such as non-insulin-dependent diabetic mellitus, atherosclerosis, hyperlipidemia, and hypertension. The topical use of ginseng roots to treat skin complaints including atopic suppurative dermatitis, wounds, and inflammation is also described in ancient Chinese texts; however, there have been relatively few studies in this area. In the present paper, we describe introduce the biological and pharmacological effects of ginsenoside Rb1 isolated from Red ginseng roots on skin damage caused by burn-wounds using male Balb/c mice (in vivo and by ultraviolet B irradiation using male C57BL/6J and albino hairless (HR-1 mice (in vivo. Furthermore, to clarify the mechanisms behind these pharmacological actions, human primary keratinocytes and the human keratinocyte cell line HaCaT were used in experiments in vitro.

  19. RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

    Science.gov (United States)

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-01-01

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

  20. Frequent disruption of the RB1 pathway in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Kania, P W; Ino, Y

    2000-01-01

    In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data fr...

  1. Brain Transport Profiles of Ginsenoside Rb1 by Glucose Transporter 1: In Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Yu-Zhu Wang

    2018-04-01

    Full Text Available Ginsenoside Rb1 (Rb1 has been demonstrated its protection for central nervous system and is apparently highly distributed to the brain. The objective of this study was to characterize Rb1 transport at the blood–brain barrier (BBB using primary cultured rat brain microvascular endothelial cells (rBMEC, an in vitro BBB model. The initial uptake velocity of Rb1 in rBMEC was temperature- and concentration-dependent, and was significantly reduced by phloretin, an inhibitor of GLUT1 transporter, but was independent of metabolic inhibitor. Furthermore, the transport of Rb1 into rBMEC was significantly diminished in the presence of natural substrate α-D-glucose, suggesting a facilitated transport of Rb1 via GLUT1 transporter. The impact of GLUT1 on the distribution of Rb1 between brain and plasma was studied experimentally in rats. Administration of phloretin (5 mg/kg, i.v. to normal rats for consecutive 1 week before Rb1 (10 mg/kg, i.v. at 0.5, 2, and 6 h did not alter Rb1 concentrations in plasma, but resulted in significant decreased brain concentrations of Rb1 compared to in the phloretin-untreated normal rats (489.6 ± 58.3 versus 105.1 ± 15.1 ng/g, 193.8 ± 11.1 versus 84.8 ± 4.1 ng/g, and 114.2 ± 24.0 versus 39.9 ± 4.9 ng/g, respectively. The expression of GLUT1 in the phloretin-treated group by western blotting analysis in vitro and in vivo experiments was significantly decreased, indicating that the decreased transport of Rb1 in brain was well related to the down-regulated function and level of GLUT1. Therefore, our in vitro and in vivo results indicate that the transport of Rb1 at the BBB is at least partly mediated by GLUT1 transporter.

  2. ATM/RB1 mutations predict shorter overall survival in urothelial cancer.

    Science.gov (United States)

    Yin, Ming; Grivas, Petros; Emamekhoo, Hamid; Mendiratta, Prateek; Ali, Siraj; Hsu, JoAnn; Vasekar, Monali; Drabick, Joseph J; Pal, Sumanta; Joshi, Monika

    2018-03-30

    Mutations of DNA repair genes, e.g. ATM/RB1 , are frequently found in urothelial cancer (UC) and have been associated with better response to cisplatin-based chemotherapy. Further external validation of the prognostic value of ATM/RB1 mutations in UC can inform clinical decision making and trial designs. In the discovery dataset, ATM/RB1 mutations were present in 24% of patients and were associated with shorter OS (adjusted HR 2.67, 95% CI, 1.45-4.92, p = 0.002). There was a higher mutation load in patients carrying ATM/RB1 mutations (median mutation load: 6.7 versus 5.5 per Mb, p = 0.072). In the validation dataset, ATM/RB1 mutations were present in 22.2% of patients and were non-significantly associated with shorter OS (adjusted HR 1.87, 95% CI, 0.97-3.59, p = 0.06) and higher mutation load (median mutation load: 8.1 versus 7.2 per Mb, p = 0.126). Exome sequencing data of 130 bladder UC patients from The Cancer Genome Atlas (TCGA) dataset were analyzed as a discovery cohort to determine the prognostic value of ATM/RB1 mutations. Results were validated in an independent cohort of 81 advanced UC patients. Cox proportional hazard regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence interval (CI) to compare overall survival (OS). ATM/RB1 mutations may be a biomarker of poor prognosis in unselected UC patients and may correlate with higher mutational load. Further studies are required to determine factors that can further stratify prognosis and evaluate predictive role of ATM/RB1 mutation status to immunotherapy and platinum-based chemotherapy.

  3. Ginsenoside Rb1 for Myocardial Ischemia/Reperfusion Injury: Preclinical Evidence and Possible Mechanisms

    Directory of Open Access Journals (Sweden)

    Qun Zheng

    2017-01-01

    Full Text Available Ginseng is an important herbal drug that has been used worldwide for many years. Ginsenoside Rb1 (G-Rb1, the major pharmacological extract from ginseng, possesses a variety of biological activities in the cardiovascular systems. Here, we conducted a preclinical systematic review to investigate the efficacy of G-Rb1 for animal models of myocardial ischemia/reperfusion injury and its possible mechanisms. Ten studies involving 211 animals were identified by searching 6 databases from inception to May 2017. The methodological quality was assessed by using the CAMARADES 10-item checklist. All the data were analyzed using RevMan 5.3 software. As a result, the score of study quality ranged from 3 to 7 points. Meta-analyses showed that G-Rb1 can significantly decrease the myocardial infarct size and cardiac enzymes (including lactate dehydrogenase, creatine kinase, and creatine kinase-MB when compared with control group (P<0.01. Significant decrease in cardiac troponin T and improvement in the degree of ST-segment depression were reported in one study (P<0.05. Additionally, the possible mechanisms of G-Rb1 for myocardial infarction are antioxidant, anti-inflammatory, antiapoptosis, promoting angiogenesis and improving the circulation. Thus, G-Rb1 is a potential cardioprotective candidate for further clinical trials of myocardial infarction.

  4. Ginsenoside Rb1 Protects Rat Neural Progenitor Cells against Oxidative Injury

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    Na Ni

    2014-03-01

    Full Text Available Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a tonic to enhance bodily functions against various ailments for hundreds of years in Far Eastern countries without apparent adverse effects. Ginsenoside Rb1, one of the most active ingredients of ginseng, has been shown to possess various pharmacological activities. Here we report that Rb1 exhibits potent neuroprotective effects against oxidative injury induced by tert-butylhydroperoxide (t-BHP. Lactate dehydrogenase (LDH assay demonstrated that incubation with 300 µm t-BHP for 2.5 h led to a significant cell loss of cultured rat embryonic cortex-derived neural progenitor cells (NPCs and the cell viability was pronouncedly increased by 24 h pretreatment of 10 µm Rb1. TUNEL staining further confirmed that pretreatment of Rb1 significantly reduced the cell apoptosis in t-BHP-induced oxidative injury. Real time PCR revealed that pretreatment with Rb1 activated Nrf2 pathway in cultured NPCs and led to an elevated expression of HO-1. The results of the present study demonstrate that Rb1 shows a potent anti-oxidative effect on cultured NPCs by activating Nrf2 pathway.

  5. Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius.

    Science.gov (United States)

    Quan, Lin-Hu; Kim, Yeon-Ju; Li, Guan Hao; Choi, Kwang-Tea; Yang, Deok-Chun

    2013-06-01

    In this study, the major ginsenoside Rb1 was transformed into the more pharmacologically active minor compound K by food grade Lactobacillus paralimentarius LH4, which was isolated from kimchi, a traditional Korean fermented food. The enzymatic reaction was analyzed by TLC, HPLC, and NMR. Using the cell-free enzyme of Lactobacillus paralimentarius LH4 at optimal conditions for 30 °C at pH 6.0, 1.0 mg ml(-1) ginsenoside Rb1 was transformed into 0.52 mg ml(-1) compound K within 72 h, with a corresponding molar conversion yield of 88 %. The cell-free enzyme hydrolyzed the two glucose moieties attached to the C-3 position and the outer glucose moiety attached to the C-20 position of the ginsenoside Rb1. The cell-free enzyme hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → gypenoside XVII and ginsenoside Rd → ginsenoside F2 → compound K. Our results indicate that Lactobacillus paralimentarius LH4 has the potential to be applied for the preparation of compound K in the food industry.

  6. Effect of enzyme on extraction of ginsenoside Rb1 and Rg3 from Panax notoginseng roots

    Science.gov (United States)

    Phuong, Nguyen Tran Xuan; Thy, Lu Thi Mong; Khang, Nguyen Luu Vinh; My, Huynh Thi Kieu; Tam, Nguyen Le Phuong; Hieu, Nguyen Huu

    2018-04-01

    Panax notoginseng is distributed throughout the north and northwest of Vietnam, especially Ha Giang, Lao Cai, and Cao Bang provinces. The root of this plant contains ginsenosides (Rb1, Rb2, Rd, Rg3), flavonoids, polyacetylene, polysaccharides, amino acids, fatty acids, and peptides. In this study, the ratios of enzyme (Viscozyme, Termamyl, Cellulase), solvent of components, and time extraction were investigated. The results showed that the highest contents of Rb1 and Rg3 were achieved in the sample extracted with the ratio of enzymes V:C:T = 1:0:0, ethanol:water (60:40, v/v) as extracting solvent in 45 minutes. Then, conditions of high performance liquid chromatography with diode array detector method to determine the content of ginsenosides Rb1 and Rg3 in the roots of Panax notoginseng were studied, including wavelength, mobile phase, and flow rate. The separation was subjected on a reversed-phase C18 column using acetonitrile (A) and water (B) as mobile phase. The gradient elution was set as follow: 0-10 min, 15-25% A; 10-20 min, 25-30% A; 20-40 min, 30-60% A; 40-60 min, 60-80% A; and 60-65 min back to 15% A before the next injection, at a flow rate of 0.5 mL/min, and the wavelength was set at 202 nm. The linear range was from 298.59 to 696.72 µg/mL for Rb1 and from 8.19 to 19.10 µg/L for Rg3. The limits of detection for Rb1 and Rg3 obtained were 0.31 µg/mL and 0.33 µg/mL, respectively. The limits of quantification were 0.95 µg/mL and 1.01 µg/mL for Rb1 and Rg3, respectively. Consequently, the high performance liquid chromatography demonstrated the highly sensitive and accurate method for determination of Rb1 and Rg3 in Panax notoginseng.

  7. Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

    Science.gov (United States)

    Yamaguchi, Y; Crane, S; Zhou, L; Ochoa, S M; Falanga, V

    2000-12-01

    Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

  8. Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling

    Directory of Open Access Journals (Sweden)

    Mao Lin

    2014-01-01

    Full Text Available Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB, which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF, and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

  9. Th1/M1 conversion to Th2/M2 responses in models of inflammation lacking cell death stimulates maturation of monocyte precursors to fibroblasts

    Directory of Open Access Journals (Sweden)

    JoAnn eTrial

    2013-09-01

    Full Text Available We have demonstrated that cardiac fibrosis arises from the differentiation of monocyte-derived fibroblasts. We present here evidence that this process requires sequential Th1 and Th2 induction promoting analogous M1 (classically activated and M2 (alternatively activated macrophage polarity. Our models are 1 mice subjected to daily repetitive ischemia reperfusion (I/R without infarction and 2 the in vitro transmigration of human mononuclear leukocytes through human cardiac microvascular endothelium. In the mouse heart, leukocytes entered after I/R in response to monocyte chemoattractant protein-1 (MCP-1 which is the major cytokine induced by this protocol. Monocytes within the heart then differentiated into fibroblasts making collagen while bearing the markers of M2 macrophages. T cells were seen in these hearts as well as in the human heart with cardiomyopathy. In the in vitro model, transmigration of the leukocytes was likewise induced by MCP-1 and some monocytes matured into fibroblasts bearing M2 markers. In this model, the MCP-1 stimulus induced a transient Th1 and M1 response that developed into a predominately Th2 and M2 response. An increase in the Th2 product IL-13 was present in both the human and the mouse models, consistent with its known role in fibrosis. In these simplified models, in which there is no cell death to stimulate an anti-inflammatory response, there is nonetheless a resolution of inflammation enabling a profibrotic environment. This induces the maturation of monocyte precursors into fibroblasts.

  10. In vitro radiosensitivity of primary human fibroblasts. Lack of correlation with acute radiation toxicity in patients with head and neck cancer

    International Nuclear Information System (INIS)

    Rudat, Volker; Dietz, Andreas; Conradt, Christian; Weber, Klaus-Josef; Flentje, Michael

    1997-01-01

    Background and purpose: There is a considerable hope among clinicians and radiobiologists to detect genetically radiosensitive patients prior to radiotherapy. A predictive assay would enable adjustment of the total irradiation dose to the individual at a constant risk of normal tissue complications. In this prospective study, the clonogenic survival assay for primary human fibroblasts to determine radiosensitivity in vitro was evaluated and then correlated with clinically observed acute radiation reactions. Materials and methods: One hundred twenty-five independent survival experiments with primary fibroblasts derived from 63 biopsies from 55 cancer and non-cancer patients were performed. Results: A wide variation of cell survival between biopsies was detected. Statistical analysis revealed a highly significantly larger interindividual than intraindividual variation of SF2 values. However, a considerable scatter of SF2 values in repeated experiments was observed in individual cases. Age, gender, disease status (cancer patient, non-cancer patient) and origin of fibroblasts (skin, periodontal tissue) were demonstrated not to be statistically significant confounding factors on the intrinsic radiosensitivity in vitro. In a prospective study, no correlation of the SF2 and acute reactions in 25 patients with head and neck cancer treated with a primary accelerated radiochemotherapy was detected. Conclusion: Our data show that the clonogenic assay is able to distinguish between intrinsic radiosensitivities of primary human fibroblasts if a statistical approach is used but does not predict acute radiation toxicity

  11. RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells.

    Science.gov (United States)

    Hesbacher, Sonja; Pfitzer, Lisa; Wiedorfer, Katharina; Angermeyer, Sabrina; Borst, Andreas; Haferkamp, Sebastian; Scholz, Claus-Jürgen; Wobser, Marion; Schrama, David; Houben, Roland

    2016-05-31

    The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.

  12. Inoculation of Pichia kudriavzevii RB1 degrades the organic acids present in raw compost material and accelerates composting.

    Science.gov (United States)

    Nakasaki, Kiyohiko; Araya, Shogo; Mimoto, Hiroshi

    2013-09-01

    In this study, the yeast strain Pichia kudriavzevii RB1 was used as an inoculum to accelerate organic matter degradation of rabbit food with added organic acids, which was used as a model food waste for composting. The RB1 strain rapidly degraded the organic acids present in the raw compost material, leading to an increase in pH beyond the neutral level, within 2 days. Both mesophilic and thermophilic bacteria proliferated faster in the compost with RB1 inoculation than in that without inoculation. Although the yeast died with the increase in compost temperature, it affected the early stages of composting prior to the thermophilic stage and accelerated the composting process by 2 days by eliminating the initial lag phase seen in the growth of other microorganisms. Moreover, populations of Bacillus thermoamylovorans, Bacillus foraminis, and Bacillus coagulans became dominant during the thermophilic stages of both composting with and without RB1 inoculation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Transformation and scattering activities of the receptor tyrosine kinase RON/Stk in rodent fibroblasts and lack of regulation by the jaagsiekte sheep retrovirus receptor, Hyal2

    International Nuclear Information System (INIS)

    Miller, A Dusty; Van Hoeven, Neal S; Liu, Shan-Lu

    2004-01-01

    The envelope (Env) protein of jaagsiekte sheep retrovirus (JSRV) can transform cells in culture and is likely to be the main factor responsible for lung cancer induction by JSRV in animals. A recent report indicates that the epithelial-cell transforming activity of JSRV Env depends on activation of the cell-surface receptor tyrosine kinase Mst1r (called RON for the human and Stk for the rodent orthologs). In the immortalized line of human epithelial cells used (BEAS-2B cells), the virus receptor Hyal2 was found to bind to and suppress the activity of RON. When Env was expressed it bound to Hyal2 causing its degradation, release of RON activity from Hyal2 suppression, and activation of pathways resulting in cell transformation. Due to difficulty with reproducibility of the transformation assay in BEAS-2B cells, we have used more tractable rodent fibroblast models to further study Hyal2 modulation of RON/Stk transforming activity and potential effects of Hyal2 on RON/Stk activation by its natural ligand, macrophage stimulating protein (MSP). We did not detect transformation of NIH 3T3 cells by plasmids expressing RON or Stk, but did detect transformation of 208F rat fibroblasts by these plasmids at a very low rate. We were able to isolate 208F cell clones that expressed RON or Stk and that showed changes in morphology indicative of transformation. The parental 208F cells did not respond to MSP but 208F cells expressing RON or Stk showed obvious increases in scattering/transformation in response to MSP. Human Hyal2 had no effect on the basal or MSP-induced phenotypes of RON-expressing 208F cells, and human, mouse or rat Hyal2 had no effect on the basal or MSP-induced phenotypes of Stk-expressing 208F cells. We have shown that RON or Stk expression in 208F rat fibroblasts results in a transformed phenotype that is enhanced by addition of the natural ligand for these proteins, MSP. Hyal2 does not directly modulate the basal or MSP-induced RON/Stk activity, although it

  14. Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

    Science.gov (United States)

    McBrayer, Samuel K; Olenchock, Benjamin A; DiNatale, Gabriel J; Shi, Diana D; Khanal, Januka; Jennings, Rebecca B; Novak, Jesse S; Oser, Matthew G; Robbins, Alissa K; Modiste, Rebecca; Bonal, Dennis; Moslehi, Javid; Bronson, Roderick T; Neuberg, Donna; Nguyen, Quang-De; Signoretti, Sabina; Losman, Julie-Aurore; Kaelin, William G

    2018-04-17

    Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

  15. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

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    Yesenia Ríos

    2011-01-01

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

  16. Optimization of lipase-catalyzed synthesis of ginsenoside Rb1 esters using response surface methodology.

    Science.gov (United States)

    Hu, Jiang-Ning; Lee, Jeung-Hee; Zhu, Xue-Mei; Shin, Jung-Ah; Adhikari, Prakash; Kim, Jae-Kyung; Lee, Ki-Teak

    2008-11-26

    In the lipase (Novozyme 435)-catalyzed synthesis of ginsenoside Rb1 esters, different acyl donors were found to affect not only the degree of conversion but also the regioselectivity. The reaction of acyl donors with short carbon chain was more effective, showing higher conversion than those with long carbon chain. Among the three solvent systems, the reaction in tert-amyl alcohol showed the highest conversion rate, while the reaction in the mixed solvent of t-BuOH and pyridine (1:1) had the lowest conversion rate. To allow the increase of GRb1 lipophilicity, we decided to further study the optimal condition of synthesis of GRb1 with vinyl decanoate with 10 carbon chain fatty acids in tert-amyl alcohol. Response surface methodology (RSM) was employed to optimize the synthesis condition. From the ridge analysis with maximum responses, the maximum GRb1 conversion was predicted to be 61.51% in a combination of factors (40.2 h, 52.95 degrees C, substrate mole ratio 275.57, and enzyme amount 39.81 mg/mL). Further, the adequacy of the predicted model was examined by additional independent experiments at the predicted maximum synthesis conditions. Results showed that the RSM was effective to optimize a combination of factors for lipase-catalyzed synthesis of ginsenoside Rb1 with vinyl decanoate.

  17. Influence of B-Complex Vitamins on the Pharmacokinetics of Ginsenosides Rg1, Rb1, and Ro After Oral Administration.

    Science.gov (United States)

    Zheng, Peihe; Chen, Yinbin; Fu, Yangyang; Wang, Hecheng; Wang, Jia; Zheng, Siwen; Xiao, Shengyuan; Wang, Yingping

    2017-11-01

    After cultivation of ginseng, ginsenosides, which are the major active ingredients of gingeng, were approved for use by the food industry, and began to be used as added functional ingredients to try to improve the quality and price of functional foods. However, the interaction between different types of ginsenosides and nutrients needs further study. We investigated the effect of B-complex vitamins (which are essential nutrients) on the pharmacokinetics of the ginsenosides protopanaxatriol-type saponin Rg 1 , protopanaxadiol-type saponin Rb 1 , and oleanolic acid-type saponin Ro after oral administration. Ginsenosides Rg 1 , Rb 1 , and Ro, with or without B-complex vitamins, respectively, were administered orally to rats to evaluate their pharmacokinetics. The concentration of ginsenosides in plasma was determined by liquid chromatography/tandem mass spectrometry. Pharmacokinetic parameters were fitted using WinNonlin v6.2. After oral coadministration with B-complex vitamins, the area under the concentration-time curve from zero to infinity (AUC 0-∞ ) of ginsenoside Rg 1 was reduced by 70%, that of ginsenoside Rb 1 was reduced by 43%, and that of ginsenoside Ro was reduced by 34%. The AUC 0-∞ of ginsenosides Rg 1 and Rb 1 showed significant differences between different treatments, but the AUC 0-∞ of ginsenoside Ro did not. These results suggest significant ginsenoside-nutrient interactions between ginsenosides Rg 1 , Rb 1 , and B-complex vitamins.

  18. Neuroprotective effects of ginsenoside Rb1 on high glucose-induced neurotoxicity in primary cultured rat hippocampal neurons.

    Science.gov (United States)

    Liu, Di; Zhang, Hong; Gu, Wenjuan; Liu, Yuqin; Zhang, Mengren

    2013-01-01

    Ginsenoside Rb1 is one of the main active principles in traditional herb ginseng and has been reported to have a wide variety of neuroprotective effects. Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, so the present study aimed to observe the effects of ginsenoside Rb1 on ER stress signaling pathways in high glucose-treated hippocampal neurons. The results from MTT, TUNEL labeling and Annexin V-FITC/PI/Hoechst assays showed that incubating neurons with 50 mM high glucose for 72 h decreased cell viability and increased the number of apoptotic cells whereas treating neurons with 1 μM Rb1 for 72 h protected the neurons against high glucose-induced cell damage. Further molecular mechanism study demonstrated that Rb1 suppressed the activation of ER stress-associated proteins including protein kinase RNA (PKR)-like ER kinase (PERK) and C/EBP homology protein (CHOP) and downregulation of Bcl-2 induced by high glucose. Moreover, Rb1 inhibited both the elevation of intracellular reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential induced by high glucose. In addition, the high glucose-induced cell apoptosis, activation of ER stress, ROS accumulation and mitochondrial dysfunction can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA) and anti-oxidant N-acetylcysteine(NAC). In conclusion, these results suggest that Rb1 may protect neurons against high glucose-induced cell injury through inhibiting CHOP signaling pathway as well as oxidative stress and mitochondrial dysfunction.

  19. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation.

    Science.gov (United States)

    Harms, Paul W; Collie, Angela M B; Hovelson, Daniel H; Cani, Andi K; Verhaegen, Monique E; Patel, Rajiv M; Fullen, Douglas R; Omata, Kei; Dlugosz, Andrzej A; Tomlins, Scott A; Billings, Steven D

    2016-03-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes

  20. Next Generation Sequencing of Cytokeratin 20-Negative Merkel Cell Carcinoma Reveals Ultraviolet Signature Mutations and Recurrent TP53 and RB1 Inactivation

    Science.gov (United States)

    Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.

    2016-01-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping

  1. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200.

    Science.gov (United States)

    Morselli, Eugenia; Shen, Shensi; Ruckenstuhl, Christoph; Bauer, Maria Anna; Mariño, Guillermo; Galluzzi, Lorenzo; Criollo, Alfredo; Michaud, Mickael; Maiuri, Maria Chiara; Chano, Tokuhiro; Madeo, Frank; Kroemer, Guido

    2011-08-15

    The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

  2. Analysis of the RB1 gene in children with retinoblastoma having residential connections to West Cumbria, England

    Energy Technology Data Exchange (ETDEWEB)

    Cowell, J K [Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Morris, J A [Department of Pathology, Royal Lancaster Infirmary, Lancaster LA1 4RP (United Kingdom); Tawn, E J [Westlakes Research Institute, Moor Row, Cumbria CA24 3JY (United Kingdom)

    2005-03-01

    Six of eight cases of retinoblastoma previously identified as having a residential association with West Cumbria, England, in which the Sellafield nuclear installation is situated, were examined for the presence of a constitutional RB1 mutation. No mutations were detected, thus providing strong evidence against an environmental or occupational genotoxic effect causing germline mutations in the parents of these children. (note)

  3. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  4. Heritable factors for radiation-induced osteosarcoma and the role of Rb1 in telomere maintenance in mesenchymal stem cells

    International Nuclear Information System (INIS)

    Rosemann, M.; Gonzalez-Vasconcellos, I.; Atkinson, M. J.

    2013-01-01

    Full text: Secondary tumors following a pediatric radiotherapy become an ever growing concern, a side effect of the improved therapeutic success leading to extended life span of the patients. Osteosarcoma (OS) and other soft-tissue sarcomas arise over proportionally frequent in the radiation field of former radiooncology patients. In an effort to map and identify inherited factors that govern individual susceptibility for these secondary, therapy associated tumors, we developed a mouse model that after injection of 227 thorium develops high numbers of OS and facilitates whole genome mapping of genetic variants that modify risk. We could identify genetic risk factors on 5 different chromosomes, that by independent segregation in the germline can interact in an additive manner and in combination alter the individual risk more than 3 fold. Using additional in-vitro studies and mouse knockout technology we could confirm that the responsible gene on the principal susceptibility locus is Rb1. Mice with a bone-specific Rb1+/- status exhibits increased bone tumor risk following irradiation. We have shown recently that the Rb1 tumor suppressor gene does not only regulates the cell cycle kinetics of mammalian cells, but that in normal osteoblasts is also crucial to protect telomeres from extensive erosion. An Rb1+/- deficiency therefore exhibit accelerated telomeric loss and, following ionising irradiation an excess of chromosomal defects. In the majority of secondary RT associated human OS, Rb1 was affected by allelic loss, whereas spontaneous human OS with no known radiation etiology show only 15%-20% Rb1 losses. It is assumed that the target cells for malignant transformation leading to OS are not the differentiating osteoblasts, but rather the long-term repopulating and pluripotent mesenchymal stem cells (MSC). These normal tissue stem cells are assumed to maintain a high degree of genome stability throughout the entire life span of an organism. One of the key factors

  5. Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

    Science.gov (United States)

    Ahn, Sung-Min; Jang, Se Jin; Shim, Ju Hyun; Kim, Deokhoon; Hong, Seung-Mo; Sung, Chang Ohk; Baek, Daehyun; Haq, Farhan; Ansari, Adnan Ahmad; Lee, Sun Young; Chun, Sung-Min; Choi, Seongmin; Choi, Hyun-Jeung; Kim, Jongkyu; Kim, Sukjun; Hwang, Shin; Lee, Young-Joo; Lee, Jong-Eun; Jung, Wang-Rim; Jang, Hye Yoon; Yang, Eunho; Sung, Wing-Kin; Lee, Nikki P; Mao, Mao; Lee, Charles; Zucman-Rossi, Jessica; Yu, Eunsil; Lee, Han Chu; Kong, Gu

    2014-12-01

    Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. © 2014 by the American Association for the Study of Liver Diseases.

  6. Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing β-Glucosidase from Aspergillus niger KCCM 11239

    Directory of Open Access Journals (Sweden)

    Kyung Hoon Chang

    2012-09-01

    Full Text Available Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu2+, Mg2+, Co2+, and acetic acid (10 mM. In the specificity tests, the enzyme was found to be active against ginsenoside Rb1, but showed very low levels of activity against Rb2, Rc, Rd, Re, and Rg1. The enzyme hydrolyzed the 20-C,β-(1→6-glucoside of ginsenoside Rb1 to generate ginsenoside Rd and Rg3, and hydrolyzed 3-C,β-(1→2-glucoside to generate F2. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds.

  7. Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

    Science.gov (United States)

    Capasso, Stefania; Alessio, Nicola; Di Bernardo, Giovanni; Cipollaro, Marilena; Melone, Mariarosa Ab; Peluso, Gianfranco; Giordano, Antonio; Galderisi, Umberto

    2014-01-01

    Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

  8. Highly Selective Bioconversion of Ginsenoside Rb1 to Compound K by the Mycelium of Cordyceps sinensis under Optimized Conditions

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    Wei-Nan Wang

    2015-10-01

    Full Text Available Compound K (CK, a highly active and bioavailable derivative obtained from protopanaxadiol ginsenosides, displays a wide variety of pharmacological properties, especially antitumor activity. However, the inadequacy of natural sources limits its application in the pharmaceutical industry. In this study, we firstly discovered that Cordyceps sinensis was a potent biocatalyst for the biotransformation of ginsenoside Rb1 into CK. After a series of investigations on the biotransformation parameters, an optimal composition of the biotransformation culture was found to be lactose, soybean powder and MgSO4 without controlling the pH. Also, an optimum temperature of 30 °C for the biotransformation process was suggested in a range of 25 °C–50 °C. Then, a biotransformation pathway of Rb1 → Rd → F2 → CK was established using high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS. Our results demonstrated that the molar bioconversion rate of Rb1 to CK was more than 82% and the purity of CK produced by C. sinensis under the optimized conditions was more than 91%. In conclusion, the combination of C. sinensis and the optimized conditions is applicable for the industrial preparation of CK for medicinal purposes.

  9. Thermodynamics, dielectric permittivity and phase diagrams of the Rb1-x(NH4xH2PO4 type proton glasses

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    S.I. Sorokov

    2010-01-01

    Full Text Available The cluster pseudospin model of proton glasses, which takes into account the energy levels of protons around the PO4 group, the long-range interactions between the hydrogen bonds, and an internal random deformational field is used to investigate thermodynamical characteristics, longitudinal and transverse dielectric permittivities of Rb1-x(ND4xD2PO4 and Rb1-x(NH4xH2AsO4 compounds. A review of experimental and theoretical works on the Rb1-x(NH4xH2PO4 type crystals is presented.

  10. PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

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    Cenciarelli, Carlo; Marei, Hany E; Felsani, Armando; Casalbore, Patrizia; Sica, Gigliola; Puglisi, Maria Ausiliatrice; Cameron, Angus J M; Olivi, Alessandro; Mangiola, Annunziato

    2016-08-16

    Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.

  11. Ginsenoside Rb1 Attenuates Agonist-Induced Contractile Response via Inhibition of Store-Operated Calcium Entry in Pulmonary Arteries of Normal and Pulmonary Hypertensive Rats

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    Rui-Xing Wang

    2015-03-01

    Full Text Available Background: Pulmonary hypertension (PH is characterized by sustained vasoconstriction, enhanced vasoreactivity and vascular remodeling, which leads to right heart failure and death. Despite several treatments are available, many forms of PH are still incurable. Ginsenoside Rb1, a principle active ingredient of Panax ginseng, exhibits multiple pharmacological effects on cardiovascular system, and suppresses monocrotaline (MCT-induced right heart hypertrophy. However, its effect on the pulmonary vascular functions related to PH is unknown. Methods: We examined the vasorelaxing effects of ginsenoside Rb1 on endothelin-1 (ET-1 induced contraction of pulmonary arteries (PAs and store-operated Ca2+ entry (SOCE in pulmonary arterial smooth muscle cells (PASMCs from chronic hypoxia (CH and MCT-induced PH. Results: Ginsenoside Rb1 elicited concentration-dependent relaxation of ET-1-induced PA contraction. The vasorelaxing effect was unaffected by nifedipine, but abolished by the SOCE blocker Gd3+. Ginsenoside Rb1 suppressed cyclopiazonic acid (CPA-induced PA contraction, and CPA-activated cation entry and Ca2+ transient in PASMCs. ET-1 and CPA-induced contraction, and CPA-activated cation entry and Ca2+ transients were enhanced in PA and PASMCs of CH and MCT-treated rats; the enhanced responses were abolished by ginsenoside Rb1. Conclusion: Ginsenoside Rb1 attenuates ET-1-induced contractile response via inhibition of SOCE, and it can effectively antagonize the enhanced pulmonary vasoreactivity in PH.

  12. Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes

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    Xu Yan

    2016-09-01

    Full Text Available Background/Aims: Ginsenoside Rb1 (GS-Rb1 is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I injuried cardiomyocytes. Materials and Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. Results: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. Conclusions: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in

  13. Ginsenoside Rb1 improves postoperative fatigue syndrome by reducing skeletal muscle oxidative stress through activation of the PI3K/Akt/Nrf2 pathway in aged rats.

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    Zhuang, Cheng-Le; Mao, Xiang-Yu; Liu, Shu; Chen, Wei-Zhe; Huang, Dong-Dong; Zhang, Chang-Jing; Chen, Bi-Cheng; Shen, Xian; Yu, Zhen

    2014-10-05

    Ginsenoside Rb1 is reported to possess anti-fatigue activity, but the mechanisms remain unknown. The aim of this study was to investigate the molecular mechanisms responsible for the anti-fatigue effect of ginsenoside Rb1 on postoperative fatigue syndrome induced by major small intestinal resection (MSIR) in aged rat. Aged rats with MSIR were administrated with ginsenoside Rb1 (15 mg/kg) once a day from 3 days before surgery to the day of sacrifice, or with saline as corresponding controls. Rats without MSIR but going through the same surgery procedure were administrated with saline as blank controls. Anti-fatigue effect was assessed by an open field test; superoxide dismutase, reactive oxygen species and malondialdehyde in skeletal muscle were determined. The mRNA levels of Akt2 and Nrf2 in skeletal muscle were measured by real-time quantitative PCR. The activation of Akt and Nrf2 was examined by western blot and immunohistofluorescence. Our results revealed that ginsenoside Rb1 significantly increased the journey and the rearing frequency, decreased the time of rest in aged rats with MSIR. In addition, ginsenoside Rb1 significantly reduced reactive oxygen species and malondialdehyde release and increased the superoxide dismutase activity of skeletal muscle in aged rats with MSIR. Ginsenoside Rb1 also increased the expression of Akt2 and Nrf2 mRNA, up-regulated Akt phosphorylation and Nrf2 nuclear translocation. These findings indicate that ginsenoside Rb1 has an anti-fatigue effect on postoperative fatigue syndrome in aged rat, and the mechanism possibly involves activation of the PI3K/Akt pathway with subsequent Nrf2 nuclear translocation and induction of antioxidant enzymes. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Pharmacokinetics and efficiency of brain targeting of ginsenosides Rg1 and Rb1 given as Nao-Qing microemulsion.

    Science.gov (United States)

    Li, Tao; Shu, Ya-Jun; Cheng, Jia-Yin; Liang, Run-Cheng; Dian, Shao-Na; Lv, Xiao-Xun; Yang, Meng-Qi; Huang, Shu-Ling; Chen, Gang; Yang, Fan

    2015-02-01

    Nao-Qing solution has been shown to be clinically effective in the treatment of acute ischemic stroke (AIS). The purpose of this study was to improve the pharmacokinetics and brain uptake of Nao-Qing, administered as an oil-in-water microemulsion. Sprague-Dawley (SD) rats were given Nao-Qing microemulsion by intranasal or intragastric routes. Samples of blood, brain, heart, liver, lung and kidney were collected at pre-determined time intervals, and the contents of ginsenosides Rg1 and Rb1 (active ingredients of the Nao-Qing microemulsion) were analyzed by high-performance liquid chromatography (HPLC). The results showed that contents of ginsenosides Rg1 and Rb1 in Nao-Qing microemulsion was 8475.13 ± 54.61 μg/ml and 6633.42 ± 527.27 μg/ml, respectively, and that the particle size, pH and viscosity of the microemulsion were 19.9 ± 5.07 nm, 6.1 and 3.056 × 10(-3 )Pas, respectively. Absorption of ginsenoside Rg1 was higher than that of ginsenoside Rb1, which was barely detectable after intragastric administration; furthermore, the concentration of ginsenoside Rg1 in blood and other tissues at each time point was lower for intragastric than for intranasal administration. Compared with intragastric administration, intranasal administration resulted in a shorter tmax (0.08 versus 1 h), a higher Cmax (16.65 versus 11.29 μg/ml), and a higher area under the concentration-time curve (AUC) (592.91 versus 101.70 μgċh/ml) in the brain. The relative rates of uptake (Re) and the ratio of peak concentration (Ce) in the brain were 126.31% and 147.48% for ginsenoside Rg1, respectively. These data illustrate that intranasal administration can promote the absorption of drugs in Nao-Qing microemulsion and achieve fast effect.

  15. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

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    Swati Tomar

    Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

  16. 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.

  17. Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

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    Xu Yan

    2014-01-01

    Full Text Available Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1 on hypoxia/ischemia (H/I injury in cardiomyocytes in vitro and the mitochondrial apoptotic pathway mediated mechanism. Methods. Neonatal rat cardiomyocytes (NRCMs for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit. Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm. Its administration also inhibited activities of caspase-9 and caspase-3. Conclusion. Administration of GS-Rb1 during H/I in vitro is involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

  18. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Jeong, Hye Gwang

    2010-01-01

    Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gβ1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gβ1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

  19. Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3

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    Kyung Hoon Chang

    2014-01-01

    Full Text Available The transformation of ginsenoside Rb1 into a specific minor ginsenoside using Aspergillus niger KCCM 11239, as well as the identification of the transformed products and the pathway via thin layer chromatography and high performance liquid chromatography were evaluated to develop a new biologically active material. The conversion of ginsenoside Rb1 generated Rd, Rg3, Rh2, and compound K although the reaction rates were low due to the low concentration. In enzymatic conversion, all of the ginsenoside Rb1 was converted to ginsenoside Rd and ginsenoside Rg3 after 24 h of incubation. The crude enzyme (β-glucosidase from A. niger KCCM 11239 hydrolyzed the β-(1→6-glucosidic linkage at the C-20 of ginsenoside Rb1 to generate ginsenoside Rd and ginsenoside Rg3. Our experimental demonstration showing that A. niger KCCM 11239 produces the ginsenoside-hydrolyzing β-glucosidase reflects the feasibility of developing a specific bioconversion process to obtain active minor ginsenosides.

  20. Study by EPR and Dielectric Constant of Proton-Glass behavior in the system Rb1-X(NH4)XH2PO4:As

    International Nuclear Information System (INIS)

    Almanza, O.; Diaz J, M.; Diaz S

    1996-01-01

    From dielectric constant and EPR measurements of the system Rb1-X(NH4)XH2PO4:As we obtained the phase-diagram Tc Vs x. EPR measurements suggest a proton-glass behavior for 0.3= =0.8. In the doping-range 0.4=< x<=1 the system shows a splitting in the low field line

  1. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

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    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  2. Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

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    Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

    2011-01-01

    Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

  3. Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study.

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    McCormick, Thaís M; Canedo, Nathalie H S; Furtado, Yara L; Silveira, Filomena A; de Lima, Roberto J; Rosman, Andréa D F; Almeida Filho, Gutemberg L; Carvalho, Maria da Glória da C

    2015-06-02

    Human papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions. Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP. HPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%). The methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was

  4. Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

    International Nuclear Information System (INIS)

    Vladusic, Tomislav; Hrascan, Reno; Pecina-Slaus, Nives; Vrhovac, Ivana; Gamulin, Marija; Franekic, Jasna; Kruslin, Bozo

    2010-01-01

    Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs. Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method. LOH of the CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component. The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior

  5. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  6. Neutron scattering investigations of the properties of the x - T phase diagram of Rb1-x(NH4)xI mixed crystals

    International Nuclear Information System (INIS)

    Smirnov, L.S.; Natkaniec, I.; Savenko, B.N.

    2002-01-01

    The x - T phase diagram of Rb 1-x (NH 4 ) x I is studied using samples with the ammonium concentration 0.01< x<0.77 over a wide temperature region of 15 to 300 K by neutron powder diffraction and inelastic incoherent neutron scattering. The results of powder diffraction studies show that at low temperatures a phase transition from α-phase to β-phase is observed at ammonium concentrations x = 0.50 and x = 0.66. Inelastic incoherent neutron scattering detects a region of the orientional glass state at ammonium concentrations 0.29< x,0.40

  7. Interpretation of the CABRI-RAFT RB1 and RB2 tests through detailed data evaluation and PAPAS-2S code analysis

    International Nuclear Information System (INIS)

    Fukano, Yoshitaka; Sato, Ikken

    2001-08-01

    The CABRI-RAFT RB1 and RB2 tests were aiming at a study on impact of fuel pin failure under an overpower condition leading to fuel melting. Using a special technique, combination of through-cladding failure and fuel melting was realized. In the RB1 test, fuel ejection was prevented under a limited fuel melting condition. On the other hand, significant fuel melting was applied in the RB2 test so as to get the fuel ejection, thereby obtaining information on the fuel ejection behavior. Interpretation for these tests through the detailed experimental data evaluation and the PAPAS-2S code analysis is performed in this study. Through this study, it is indicated that molten fuel ejection can be prevented with the low smear density fuel as far as the fuel melting is not large for a slit-type cladding defect. Fuel ejection becomes possible in the case of significant fuel melting with a very thin solid fuel shell surrounding the molten fuel cavity. However, the rapidness of the fuel ejection with the low smear density fuel is less pronounced compared with that of the high smear density fuel. It is also confirmed that there is considerable DN-precursor release into the coolant flow already before fuel ejection. The result is very useful for evaluation of anomaly detection with DN signal observation. (author)

  8. Study of ammonium molecular ion impurity modes in Rb1-x(NH4)xI mixed crystals by inelastic incoherent neutron scattering

    International Nuclear Information System (INIS)

    Smirnov, L.S.; Natkaniec, I.; Ollivier, J.; Dianoux, J.A.; Martinez Sarrion, M.L.; Mestres, L.

    2010-01-01

    The study of ammonium dynamics in Rb 1-x (NH 4 ) x I mixed crystals was carried out by inelastic incoherent neutron scattering in the concentration region of orientationally disordered α-phase, 0.0< x<0.40, at the temperature range from 2 to 150 K. The observed resonance modes correspond to three energy regions: 0.19-0.481 (I), 0.56-3.0 (II) and 4.0-10.0 (III) meV. The modes of region I could be described by rotational tunneling energies of the multipole moments of ammonium ions. The modes within energy region II correspond to the calculated rotational tunneling energies between splitted levels of the ground librational level of ammonium ion. The modes of region III can be described as local gap modes of ammonium ion because they are located between acoustic and optic branches of RbI phonon density of states

  9. Inhibition of Autophagy via Activation of PI3K/Akt Pathway Contributes to the Protection of Ginsenoside Rb1 against Neuronal Death Caused by Ischemic Insults

    Directory of Open Access Journals (Sweden)

    Tianfei Luo

    2014-09-01

    Full Text Available Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1 on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO and Monodansylcadaverine (MDC staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

  10. Carbon nanotubes as carriers of Panax ginseng metabolites and enhancers of ginsenosides Rb1 and Rg1 anti-cancer activity

    Science.gov (United States)

    Lahiani, Mohamed H.; Eassa, Souzan; Parnell, Charlette; Nima, Zeid; Ghosh, Anindya; Biris, Alexandru S.; Khodakovskaya, Mariya V.

    2017-01-01

    A major benefit to nanomaterial based-medicine is the ability to provide nanosized vehicles for sporadic metabolites. Here, we describe how the conjugation of valuable ginseng secondary metabolites (ginsenoside Rb1 or Rg1) with carbon nanotubes (CNT) can enhance their anti-proliferative and anti-cancer effects. Ginsenoside-CNT conjugate (Rb-CNT or Rg-CNT) permitted the ginsenosides to be used at a low dose, yet achieve a higher incidence of cancer killing. We were able to demonstrate that the ginsenoside-CNT conjugate can decrease cell viability up to 62% in breast cancer cells (MCF-7) and enhance antiproliferation of drug-resistant pancreatic cancer cells (PANC-1) by 61%. The interaction of the ginsenoside-CNT conjugate with breast cancer cells was studied using Raman Spectroscopy mapping. Total transcriptome profiling (Affymetrix platform) of MCF-7 cells treated with the ginsenoside-CNT conjugate shows that a number of cellular, apoptotic and response to stimulus processes were affected. Therefore, our data confirmed the potential use of CNT as a drug delivery system.

  11. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

    Science.gov (United States)

    Shaikh, Faraz; Sanehi, Parvish; Rawal, Rakesh

    2012-01-01

    Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. Abbreviations HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins. PMID:22829740

  12. Influence of concentration and temperature on tunneling and rotational dynamics of ammonium in Rb1-x (NH4)x I mixed crystals

    International Nuclear Information System (INIS)

    Natkaniec, I.; Dianoux, A.J.; Martinez-Sarrion, M.L.; Mestres, L.; Herraiz, M.; Smirnov, L.S.; Shuvalov, L.A.

    2001-01-01

    The Rb 1x (NH 4 ) x I mixed crystals are studied by inelastic incoherent neutron scattering using time-of-flight spectrometers in the concentration region of the x-T phase diagram 0.01≤x≤0.66 at 5≤T≤150K, where dynamic and static orientational disorder phases are generally found. It is shown that at 5 K rotational tunneling levels for ammonium concentrations x=0.01, 0.02 and 0.06 are similar. Additional tunneling levels are observed for x=0.16 which can be explained as the result of T-states splitting for account of NH 4 -NH 4 interaction. Tunneling levels are not observed for x=0.40 as the result of forming of orientational glass state. The elastic incoherent structure factors for concentrations 0.01≤x≤0.16 (dynamic orientational disordered α-phase), x=0.40 (orientational glass state) and 0.50≤x≤0.66 (orientational ordered state) have different temperature dependences

  13. 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.

  14. Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma.

    Science.gov (United States)

    Liu, Qingqing; Cornejo, Kristine M; Cheng, Liang; Hutchinson, Lloyd; Wang, Mingsheng; Zhang, Shaobo; Tomaszewicz, Keith; Cosar, Ediz F; Woda, Bruce A; Jiang, Zhong

    2018-04-01

    Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n = 33; RO, n = 32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. Comparative modeling and docking studies of p16ink4/Cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1

    Directory of Open Access Journals (Sweden)

    e Zahra Syeda Naqsh

    2013-01-01

    Full Text Available Abstract Background Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1 Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase (2 Growth inhibitory pathways (p53/Rb/P14ARF, STK11 (3 Apoptotic pathways (Bcl-2/Bax/Fas/FasL. Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. Results YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of −0.132 and −0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. Conclusion This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of

  16. Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie A Wallace

    Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

  17. 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.

  18. Energy brands lack vitality

    International Nuclear Information System (INIS)

    Godri, S.; Wilders, E.

    2004-01-01

    The three Dutch energy companies (Nuon, Essent and Eneco Energie) have relatively little brand strength. The brands are not perceived to be sufficiently different from one another and are not valued by consumers. With liberalisation imminent, this is hardly a strong starting point. How can you win over consumers if it is not clear what is on offer? In the business market, decision-makers are better placed to distinguish between brands. However, the brands lack vitality in this sector of the market too. The only consolation is that the situation is by no means exclusive to the Netherlands [nl

  19. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  20. Microprobe analysis of human fibroblasts

    International Nuclear Information System (INIS)

    Allan, G.L.; Zhu, J.; Legge, G.J.F.

    1985-01-01

    The Melbourne Proton Microprobe has been used to study the copper content in human skin fibroblast cells derived from patients with the genetic disease Menkes Syndrome. Both normal and diseased cells have been studied to investigate any elemental differences occurring between the two cell types. This paper details the preparatory techniques necessary for individual cell analysis and presents the elemental information with a new three dimensional contour mapping technique. These maps are used to highlight elemental differences between normal and mutant fibroblasts. The work also confirms the expected copper excess found in the Menkes cell and indicates that the microprobe can be used for rapid identification of a Menkes carrier

  1. Connective tissue fibroblasts and Tcf4 regulate myogenesis

    Science.gov (United States)

    Mathew, Sam J.; Hansen, Jody M.; Merrell, Allyson J.; Murphy, Malea M.; Lawson, Jennifer A.; Hutcheson, David A.; Hansen, Mark S.; Angus-Hill, Melinda; Kardon, Gabrielle

    2011-01-01

    Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis. PMID:21177349

  2. Case report 511: Fibroblastic rheumatism

    International Nuclear Information System (INIS)

    Hernandez, R.J.; Martel, W.; Headington, J.T.; Kaufman, R.A.; Cincinnati Univ., OH

    1989-01-01

    We report a ten-year-old child with the newly described entity of fibroblastic rheumatism. This child developed rapid, progressive, symmetrical polyarthritis, similar to the radiographic appearance of juvenile rheumatoid arthritis, except for the rapidity of progression. The polyarthritis was preceded by the development of skin nodules with characteristic histological changes. (orig./GDG)

  3. Evaluation of IL-12RB1, IL-12B, CXCR-3 and IL-17a expression in cases affected by a non-healing form of cutaneous leishmaniasis: an observational study design.

    Science.gov (United States)

    Moafi, Mohammad; Rezvan, Hossein; Sherkat, Roya; Taleban, Roya; Asilian, Ali; Zarkesh Esfahani, Seyed Hamid; Nilforoushzadeh, Mohammad Ali; Jaffary, Fariba; Feizi, Awat

    2017-01-27

    Seldom cutaneous leishmaniasis (CL) may present as a lasting and active lesion(s), known as a non-healing form of CL (NHCL). Non-functional type 1 T helper (Th1) cells are assumed the most important factor in the outcome of the disease. The present study aims to assess some molecular defects that potentially contribute to Th1 impairment in NHCL. This prospective observational study will be implemented among five groups. The first and second groups comprise patients afflicted with non-healing and healing forms of CL, respectively. The third group consists of those recovered participants who have scars as a result of CL. Those participants who have never lived or travelled to endemic areas of leishmaniasis will comprise the fourth group. The fifth group comprises participants living in hyperendemic areas for leishmaniasis, although none of them have been afflicted by CL. The aim is to recruit 10 NHCL cases and 30 participants in each of the other groups. A leishmanin skin test (LST) will be performed to assess in vivo immunity against the Leishmania infection. The cytokine profile (interleukin (IL)-12p70, interferon (IFN)-γ, C-X-C motif chemokine ligand (CXCL)-11 and IL-17a) of the isolated peripheral blood mononuclear cells (PBMCs) will be evaluated through ELISA. Real-time PCR will determine the C-X-C motif chemokine receptor (CXCR)-3 and IL-17a gene expression and expression of IL-12Rβ1 will be assessed by flow cytometry. Furthermore, IL-12B and IL-12RB1 mutation analysis will be performed. It is anticipated that the outcome of the current study will identify IL-12B and IL-12RB1 mutations, which lead to persistent lesions of CL. Furthermore, our expected results will reveal an association between NHCL and pro-inflammatory cytokines (IL-12p70, IFN-γ IL-17a and CXCL-11), as well as CXCR-3 expression. This study has been approved by a local ethical committee. The final results will be disseminated through peer-reviewed journals and scientific conferences

  4. Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

    Directory of Open Access Journals (Sweden)

    Pengfei Ge

    2013-10-01

    Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

  5. Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

    Science.gov (United States)

    Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

    2018-02-15

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

  6. 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.

  7. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  8. Radiosensitivity in cultured human fibroblasts

    International Nuclear Information System (INIS)

    Cox, R.; Masson, W.K.

    1980-01-01

    Caution is urged in the use of freshly isolated cultures of human diploid fibroblasts for quantitative studies of radiosensitivity. The distribution of x ray sensitivities of 'normal' human fibroblast cultures of foetal origin (10 subjects, skin or lung biopsy) and post-foetal origin (34 subjects, skin biopsy) are compared with the distribution in 12 patients with ataxia telangiectasia (probability of including any one of these in a normal post-foetal distribution is 0.01%). Cultures from nominally normal subjects showed a broad distribution of D 0 range of 98 +- 160 rad and assuming normal distribution, a mean +- one standard deviation of 122 +- 17 rad. Mean D 0 values for foetal origin cultures were 117 +- 12; values for post-foetal cultures D 0 were 124 +- 18. No systematic variation in D 0 was observed for age of donor, number of cell divisions in culture or for cloning efficiency. For ataxia telangiectasia D 0 values were 46 +- 7 rad. (U.K.)

  9. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Szlachcic A

    2016-08-01

    Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

  10. Impact of matrix stiffness on fibroblast function

    Energy Technology Data Exchange (ETDEWEB)

    El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

    2017-05-01

    Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

  11. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

    Directory of Open Access Journals (Sweden)

    Paul P Bonvallet

    Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

  12. Fibroblast cultures in duchenne muscular dystrophy

    International Nuclear Information System (INIS)

    Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

    1977-01-01

    Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

  13. Differences in [14C]glycerol utilization in normal and familial hypercholesterolemic fibroblasts

    International Nuclear Information System (INIS)

    Shireman, R.B.; Durieux, J.

    1991-01-01

    It is known that cultured fibroblasts from familial hypercholesterolemia (FH) patients lack the normal cell receptor for low density lipoprotein (LDL) and that the absence of receptor-mediated transport of LDL cholesterol into these cells results in increased cellular synthesis of cholesterol. After 20 h perincubation in lipid-free medium, cultured FH fibroblasts incorporated significantly greater amounts of [ 14 C]glycerol into cellular lipids than did normal fibroblasts. Relative to the control medium which contained only bovine serum albumin (BSA), preincubation with 5% fetal bovine serum or 50 micrograms LDL/ml decreased [ 14 C]glycerol incorporation by both cell types. FH cells utilized more [ 14 C]glycerol for phospholipid synthesis and less for triglyceride synthesis than normal cells. This study indicates that LDL may be important in the transport of glycerides, as well as cholesterol, to cells

  14. Fibroblasts in fibrosis: novel roles and mediators

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Kendall

    2014-05-01

    Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

  15. Mycophenolic acid suppresses human pterygium and normal tenon fibroblast proliferation in vitro.

    Science.gov (United States)

    Amer, Radgonde; Rabinowich, Liane; Maftsir, Genia; Puxeddu, Ilaria; Levi-Schaffer, Francesca; Solomon, Abraham

    2010-10-01

    To investigate whether mycophenolic acid (MPA) exerts antifibrotic effects on pterygium fibroblasts (PFB) with and without stimulation with fibrogenic cytokines, and to compare the efficacy of MPA with mitomycin (MMC) and dexamethasone (DXM) on PFB and tenon fibroblasts (TFB). TFB and PFB were obtained from tissue explants during strabismus or pterygium surgery. Proliferation of subconfluent fibroblasts ± basic fibroblast growth factor (bFGF) (10 ng/ml) was assessed by using the (3H) thymidine-incorporation assay. Cell cultures were incubated with MPA, MMC or DXM. Apoptosis was evaluated by quantifying Annexin V and propidium iodide positive cells with flow cytometry. MPA showed a concentration-dependent inhibition of proliferation of PFB ± bFGF as well as TFB ± bFGF. The antiproliferative effect of MPA was comparable with that of MMC and DXM. Short exposure of PFB to MPA under profibrogenic conditions was significantly inhibitory. No apoptotic effect was found on TFB. MPA suppressed tenon and pterygium fibroblast proliferation in vitro under basal and profibrogenic conditions. It was comparable with MMC under long-term exposure, but MMC was more suppressive under short-term exposure. MPA may be safer than MMC due to a more specific mechanism of action and lack of cytotoxicity. Further investigation is warranted regarding MPA concentrations that will lead to a potent antiproliferative effect in vivo.

  16. Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

    Science.gov (United States)

    Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

    2018-01-01

    RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

  17. 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.

  18. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    International Nuclear Information System (INIS)

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-01-01

    The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

  19. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

    2017-06-15

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  20. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    International Nuclear Information System (INIS)

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-01-01

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  1. Collagen matrix as a tool in studying fibroblastic cell behavior.

    Science.gov (United States)

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.

  2. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

  3. Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

    International Nuclear Information System (INIS)

    Schaudies, R.P.; Wray, H.L.

    1988-01-01

    We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells

  4. Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

    DEFF Research Database (Denmark)

    Jeong, J; Han, I; Lim, Y

    2001-01-01

    of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C...

  5. Role of periodontal ligament fibroblasts in osteoclastogenesis: a review

    NARCIS (Netherlands)

    Sokos, D.; Everts, V.; de Vries, T.J.

    2015-01-01

    During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract

  6. Fibroblast growth factor 23 - et fosfatregulerende hormon

    DEFF Research Database (Denmark)

    Beck-Nielsen, Signe; Pedersen, Susanne Møller; Kassem, Moustapha

    2010-01-01

    Fibroblast growth factor 23 (FGF23) er et nyligt identificeret fosfatonin. FGF23's fysiologiske hovedfunktion er at opretholde normalt serumfosfat og at virke som et D-vitaminmodregulatorisk hormon. Sygdomme, der er koblet til forhøjet serum FGF23, er hypofosfatæmisk rakitis, fibrøs dysplasi og t...

  7. Cryopreservation of canine ovarian and testicular fibroblasts.

    Science.gov (United States)

    Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

    2009-01-01

    To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

  8. Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

    DEFF Research Database (Denmark)

    Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

    2018-01-01

    /LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

  9. 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.

  10. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

  11. SWAP-70 contributes to spontaneous transformation of mouse embryo fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Yu-Tzu; Shu, Chung-Li; Lai, Jing-Yang; Lin, Ching-Yu; Chuu, Chih-Pin [Institute of Cellular and System Medicine National Health Research Institute, Zhunan Town 35053, Miaoli County, Taiwan, ROC (China); Morishita, Kazuhiro; Ichikawa, Tomonaga [Division of Tumor and Cellular Biochemistry Department of Medical Sciences Faculty of Medicine University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki-shi, Miyazaki 889-1692 Japan (Japan); Jessberger, Rolf [Faculty of Medicine Carl Gustav Carus, Institute of Physiological Chemistry, Dresden University of Technology, Dresden (Germany); Fukui, Yasuhisa, E-mail: 990412@nhri.org.tw [Institute of Cellular and System Medicine National Health Research Institute, Zhunan Town 35053, Miaoli County, Taiwan, ROC (China)

    2016-07-15

    Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, a putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs. - Highlights: • Mouse embryo fibroblasts (MEFs) lacking SWAP-70 do not cause spontaneous transform. • Adding back of SWAP-70 to SWAP-70-deficient MEFs induces spontaneous transformation. • SWAP-70 is required for spontaneous transformation of MEFs.

  12. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis.

    Science.gov (United States)

    Pardo, Annie; Selman, Moisés

    2016-12-01

    Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible lung disease of unknown etiology, elusive pathogenesis, and very limited therapeutic options. The hallmarks of IPF are aberrant activation of alveolar epithelial cells and accumulation of fibroblasts and myofibroblasts along with excessive production of extracellular matrix. The linkage of aging with this disorder is uncertain, but a number of changes associated with aging, including telomere attrition, cell senescence, and mitochondrial dysfunction, have been revealed in IPF lungs. Also, aging seems to confer a profibrotic phenotype upon fibroblasts and to increase the severity of the fibrogenic response in non-IPF fibrotic lung disorders. Better knowledge of the pathophysiological mechanisms linking aging to IPF will advance understanding of its pathogenesis and may provide new therapeutic windows to treatment of this devastating disease.

  13. Triptonide inhibits the pathological functions of gastric cancer-associated fibroblasts.

    Science.gov (United States)

    Wang, Zhenfei; Ma, Daguang; Wang, Changshan; Zhu, Zhe; Yang, Yongyan; Zeng, Fenfang; Yuan, Jianlong; Liu, Xia; Gao, Yue; Chen, Yongxia; Jia, Yongfeng

    2017-12-01

    Direct attacks on tumour cells with chemotherapeutic drugs have the drawbacks of accelerating tumour metastasis and inducing tumour stem cell phenotypes. Inhibition of tumour-associated fibroblasts, which provide nourishment and support to tumour cells, is a novel and promising anti-tumour strategy. However, effective drugs against tumour-associated fibroblasts are currently lacking. In the present study, we explored the possibility of inhibiting the pathological functions of tumour-associated fibroblasts with triptonide. Paired gastric normal fibroblasts (GNFs) and gastric cancer-associated fibroblasts (GCAFs) were obtained from resected tissues. GCAFs showed higher capacities to induce colony formation, migration, and invasion of gastric cancer cells than GNFs. Triptonide treatment strongly inhibited the colony formation-, migration-, and invasion-promoting capacities of GCAFs. The expression of microRNA-301a was higher and that of microRNA-149 was lower in GCAFs than in GNFs. Triptonide treatment significantly down-regulated microRNA-301a expression and up-regulated microRNA-149 expression in GCAFs. Re-establishment of microRNA expression balance increased the production and secretion of tissue inhibitor of metalloproteinase 2, a tumour suppressive factor, and suppressed the production and secretion of IL-6, an oncogenic factor, in GCAFs. Moreover, triptonide treatment abolished the ability of GCAFs to induce epithelial-mesenchymal transition in gastric cancer cells. These results indicate that triptonide inhibits the malignancy-promoting capacity of GCAFs by correcting abnormalities in microRNA expression. Thus, triptonide is a promisingly therapeutic agent for gastric cancer treatment, and traditional herbs may be a valuable source for developing new drugs that can regulate the tumour microenvironment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Gene targeting in adult rhesus macaque fibroblasts

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2008-03-01

    Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

  15. A Transcriptomic and Epigenomic Comparison of Fetal and Adult Human Cardiac Fibroblasts Reveals Novel Key Transcription Factors in Adult Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Malin K.B. Jonsson, PhD

    2016-12-01

    Full Text Available Cardiovascular disease remains the number one global cause of death and presents as multiple phenotypes in which the interplay between cardiomyocytes and cardiac fibroblasts (CFs has become increasingly highlighted. Fetal and adult CFs influence neighboring cardiomyocytes in different ways. Thus far, a detailed comparison between the two is lacking. Using a genome-wide approach, we identified and validated 2 crucial players for maintaining the adult primary human CF phenotype. Knockdown of these factors induced significant phenotypical changes, including senescence and reduced collagen gene expression. These may now represent novel therapeutic targets against deleterious functions of CFs in adult cardiovascular disease.

  16. DNA rearrangements from γ-irradiated normal human fibroblasts preferentially occur in transcribed regions of the genome

    International Nuclear Information System (INIS)

    Forrester, H.B.; Radford, I.R.

    2003-01-01

    Full text: DNA rearrangement events leading to chromosomal aberrations are central to ionizing radiation-induced cell death. Although DNA double-strand breaks are probably the lesion that initiates formation of chromosomal aberrations, little is understood about the molecular mechanisms that generate and modulate DNA rearrangement. Examination of the sequences that flank sites of DNA rearrangement may provide information regarding the processes and enzymes involved in rearrangement events. Accordingly, we developed a method using inverse PCR that allows the detection and sequencing of putative radiation-induced DNA rearrangements in defined regions of the human genome. The method can detect single copies of a rearrangement event that has occurred in a particular region of the genome and, therefore, DNA rearrangement detection does not require survival and continued multiplication of the affected cell. Ionizing radiation-induced DNA rearrangements were detected in several different regions of the genome of human fibroblast cells that were exposed to 30 Gy of γ-irradiation and then incubated for 24 hours at 37 deg C. There was a 3- to 5-fold increase in the number of products amplified from irradiated as compared with control cells in the target regions 5' to the C-MYC, CDKN1A, RB1, and FGFR2 genes. Sequences were examined from 121 DNA rearrangements. Approximately half of the PCR products were derived from possible inter-chromosomal rearrangements and the remainder were from intra-chromosomal events. A high proportion of the sequences that rearranged with target regions were located in genes, suggesting that rearrangements may occur preferentially in transcribed regions. Eighty-four percent of the sequences examined by reverse transcriptase PCR were from transcribed sequences in IMR-90 cells. The distribution of DNA rearrangements within the target regions is non-random and homology occurs between the sequences involved in rearrangements in some cases but is not

  17. 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)

  18. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    OpenAIRE

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; ...

  19. 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...

  20. Immortalization of Werner syndrome and progeria fibroblasts

    International Nuclear Information System (INIS)

    Saito, H.; Moses, R.E.

    1991-01-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

  1. Coupling of cytoskeleton functions for fibroblast locomotion

    DEFF Research Database (Denmark)

    Couchman, J R; Lenn, M; Rees, D A

    1985-01-01

    caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

  2. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...

  3. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  4. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  5. Toll-like receptor 9 mediated responses in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Ingrid Kristine Ohm

    Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

  6. Fibroblast Senescence and Squamous Cell Carcinoma: How wounding therapies could be protective

    Science.gov (United States)

    Travers, Jeffrey B.; Spandau, Dan F; Lewis, Davina A.; Machado, Christiane; Kingsley, Melanie; Mousdicas, Nico; Somani, Ally-Khan

    2014-01-01

    Background Squamous cell carcinoma (SCC), which has one of the highest incidences of all cancers in the United States, is an age-dependent disease as the majority of these cancers are diagnosed in people over 70 years of age. Recent findings have led to a new hypothesis on the pathogenesis of SCC. Objectives To evaluate the potential of preventive therapies to reduce the incidence of SCC in at-risk geriatric patients. Materials and Methods Survey of current literature on wounding therapies to prevent SCCs. Results This new hypothesis of SCC photocarcinogenesis states that senescent fibroblasts accumulate in geriatric dermis resulting in a reduction in dermal insulin-like growth factor-1 (IGF-1) expression. This lack of IGF-1 expression sensitizes epidermal keratinocytes to fail to suppress UVB-induced mutations leading to increased proclivity to photocarcinogenesis. Recent evidence suggests that dermal wounding therapies, specifically dermabrasion and fractionated laser resurfacing, can decrease the proportion of senescent dermal fibroblasts, increase dermal IGF-1 expression, and correct the inappropriate UVB response found in geriatric skin, thus protecting geriatric keratinocytes from UVB-induced SCC initiation. Conclusions In this review, we will discuss the translation of pioneering basic science results implicating commonly used dermal fibroblast rejuvenation procedures as preventative treatments for SCC. PMID:23437969

  7. Myogenic conversion of bladder fibroblasts by construction and ...

    African Journals Online (AJOL)

    The cultured primary bladder fibroblasts were transfected by pEGFP-Myod1 with Lipofection 2000 reagent. The results showed that expression of Myod1 could cause myogenic differentiation of bladder fibroblasts. These findings support the possibility of an alternative approach to exploit the capacity of Myod1 to activate ...

  8. 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.

  9. 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.

  10. Myxoinflammatory fibroblastic sarcoma: spectrum of disease and imaging presentation

    Energy Technology Data Exchange (ETDEWEB)

    Gaetke-Udager, Kara; Yablon, Corrie M.; Morag, Yoav [University of Michigan Health System, Department of Radiology, Ann Arbor, MI (United States); Lucas, David R. [University of Michigan Health System, Department of Pathology, Ann Arbor, MI (United States)

    2016-03-15

    To describe the imaging findings of a series of myxoinflammatory fibroblastic sarcomas (MFSs) from our institution, including a case of dedifferentiated MFS and two cases with areas of high-grade tumor, in addition to typical cases of low-grade tumor. To correlate the imaging findings with the pathologic features of these tumors. IRB approval was obtained. Retrospective search of the pathology database at our institution from 2000 to 2015 identified seven cases of MFS with available imaging. Imaging, pathology, and clinical data were reviewed. Unlike the majority of well-differentiated tumors in our series (four cases), one tumor showed dedifferentiation and two cases had areas of high-grade tumor. The dedifferentiated tumor showed peripheral post-contrast enhancement. One case with a substantial high-grade component showed osseous destruction and peripheral enhancement in the high-grade area, while the low-grade component enhanced diffusely. The second case had a small high-grade area and showed diffuse enhancement. All three of these cases had non-acral locations and lacked association with a tendon. The four cases of low-grade MFS demonstrated diffuse enhancement, were located in the distal extremities, and were associated with a tendon. The imaging findings of dedifferentiated and high-grade MFS differ from the more typical low-grade tumors in that they have nonenhancing areas, a non-acral location, lack association with a tendon, and may involve bone. The radiologist should be aware that MFS represents a spectrum that includes low-grade tumors, tumors with high-grade areas, and tumors with dedifferentiation and that this spectrum presents with differing imaging features. (orig.)

  11. Collagen expression in fibroblasts with a novel LMNA mutation

    International Nuclear Information System (INIS)

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy

  12. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

    Science.gov (United States)

    Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

    2016-01-01

    Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

  13. Matrix-directed differentiation of human adipose-derived mesenchymal stem cells to dermal-like fibroblasts that produce extracellular matrix.

    Science.gov (United States)

    Sivan, Unnikrishnan; Jayakumar, K; Krishnan, Lissy K

    2016-10-01

    Commercially available skin substitutes lack essential non-immune cells for adequate tissue regeneration of non-healing wounds. A tissue-engineered, patient-specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose-derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal-like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast-conditioned medium and growth factors, were used as a fibroblast-specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast-specific protein-1 (FSP-1) and a panel of ECM molecules specific to the dermis, such as fibrillin-1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal-specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal-like fibroblasts for regenerative medicine, a matrix-directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Doubling potential of fibroblasts from different species after ionising radiation

    International Nuclear Information System (INIS)

    Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

    1976-01-01

    It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerates acquisition of this potential; human fibroblasts showed an intermediate response, since ionising radiation neither established the cultures as with mouse cells nor reduced the number of cells produced as with chicken fibroblasts. Possible explanations for the different behaviour of the species are offered. (U.K.)

  15. Ethnic variations of a retinoblastoma susceptibility gene (RB1 ...

    Indian Academy of Sciences (India)

    Unknown

    1Department of Paediatrics, National University of Singapore, Lower Kent Ridge Road, Singapore 119074. 2National Neuroscience Institute, Singapore 308433. 3Department of Paediatrics, University Diponegoro, Indonesia 50241. 4Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA.

  16. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    Choi, Sang Wook; Ham, Yong Hoh; Kim, Mee Heui

    1994-04-01

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  17. 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

  18. 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.

  19. 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.

  20. Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

    Science.gov (United States)

    Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

    2017-10-01

    Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

  1. Matrix metalloproteinase inhibition reduces contraction by dupuytren fibroblasts.

    Science.gov (United States)

    Townley, William A; Cambrey, Alison D; Khaw, Peng T; Grobbelaar, Adriaan O

    2008-11-01

    Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

  2. Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

    Science.gov (United States)

    Nakagawa, S; Pawelek, P; Grinnell, F

    1989-06-01

    To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

  3. Alzheimer skin fibroblasts show increased susceptibility to free radicals.

    Science.gov (United States)

    Tesco, G; Latorraca, S; Piersanti, P; Piacentini, S; Amaducci, L; Sorbi, S

    1992-11-01

    We have studied the response to toxic oxygen metabolites of fibroblasts derived from skin biopsies of 5 patients with familial (FAD) and 4 with sporadic (AD) Alzheimer's disease compared with those derived from 4 normal controls. Fibroblasts were damaged by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by 50 munits of xanthine-oxidase (Xo). To quantify cell damage we measured lactate dehydrogenase (LDH) activity in the culture medium and cell viability in fibroblast cultures. We found a significant increase in LDH activity in the FAD vs. controls and also in the AD vs. controls.

  4. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Story, M.T.

    1989-01-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

  5. A Phenomenological Study on Lack of Motivation

    Science.gov (United States)

    Educational Research and Reviews, 2013

    2013-01-01

    The aim of this research is to point out the underlying reasons about the lack of motivation at academic activities concerning Attribution Theory. Attribution Theory trys to understand how the people answer "why" question and how they do casual explanations. This research is a qualitative based research. It used the phenomenological…

  6. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

    2011-08-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

  7. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

    2011-01-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

  8. The radiation response of human dermal fibroblasts

    Science.gov (United States)

    Mitchell, Stephen Andrew

    A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: puse of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration

  9. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Science.gov (United States)

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant. PMID:25054322

  10. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Directory of Open Access Journals (Sweden)

    Hongbo Wei

    2014-07-01

    Full Text Available Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant.

  11. Relationship among expression of basic-fibroblast growth factor ...

    African Journals Online (AJOL)

    Relationship among expression of basic-fibroblast growth factor, MTDH/Astrocyte elevated gene-1, adenomatous polyposis coli, matrix metalloproteinase 9,and COX-2 markers with prognostic factors in prostate carcinomas.

  12. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  13. Factor XIIIa is expressed by fibroblasts in fibrovascular tumors.

    Science.gov (United States)

    Nemeth, A J; Penneys, N S

    1989-10-01

    Factor XIIIa (FXIIIa), a blood and intracellularly produced coagulation factor, has been found in a variety of cell types including fibroblast-like mesenchymal cells, and has been shown to stimulate the proliferation of fibroblasts and some neoplastic cells in vitro. We have already shown that the dendritic fibroblasts composing the fibrous papule contain this factor. We hypothesized that histopathologically similar fibrovascular tumors may also express FXIIIa and, in this report, show that the large stellate fibroblasts found in acquired digital fibrokeratomas, angiofibromas (adenoma sebaceum of Pringle), and oral fibroma (oral fibrous hyperplasia) also express FXIIIa. We postulate that FXIIIa, possibly acting as a growth factor, may be a common denominator in the pathogenesis of these tumors. Another possibility is that these tumors may be the consequence of a local overproduction of FXIIIa in response to an, as yet, unidentified stimulus.

  14. HGF is released from buccal fibroblasts after smokeless tobacco stimulation

    DEFF Research Database (Denmark)

    Dabelsteen, S; Christensen, S; Gron, B

    2005-01-01

    on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells......To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w....... Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions....

  15. Transient expression of acidic fibroblast growth factor in pea (Pisum ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... a 100 W, long-wave UV lamp (Black Ray model B-100 AP; Ultra- ... frequency) was used to estimate the treatment efficiency during 15 days post .... Crystal structure of fibroblast growth factor receptor ectodomain bound to.

  16. The effect of tetraethylammonium on intracellular calcium concentration in Alzheimer's disease fibroblasts with APP, S182 and E5-1 missense mutations.

    Science.gov (United States)

    Failli, P; Tesco, G; Ruocco, C; Ginestroni, A; Amaducci, L; Giotti, A; Sorbi, S

    1996-04-26

    It has been proposed that the lack of intracellular calcium concentration ([Ca2+]i) increase induced by the potassium channel blocker tetraethylammonium (TEA) in skin fibroblast cell lines identifies patients with both sporadic and familial Alzheimer's disease (AD). In order to verify this hypothesis, the effect of TEA on [Ca2+]i was studied in single fura-2-loaded skin fibroblast cell lines available in the Tissue Bank of the Italian Research Council. Four out of eight familial AD patients (one patient with S182 mutation, one patient with E5-1 mutation and two patients with 717 Val-->Ile APP mutation) and two out of five sporadic AD patients showed a positive response to TEA, whereas five out of 11 control lines were unresponsive. Our data suggest that the absence of the TEA-induced increase in [Ca2+]i in skin fibroblast cell lines does not identify all AD patients.

  17. Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

    Science.gov (United States)

    Sasaki, Norihiko; Itakura, Yoko; Toyoda, Masashi

    2017-04-18

    Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast

  18. 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.

  19. 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.

  20. Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0512 TITLE: Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer PRINCIPAL INVESTIGATOR: Andrew...SUBTITLE 5a. CONTRACT NUMBER Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer 5b. GRANT NUMBER W81XWH-15-1-0512 5c. PROGRAM...blocked by the addition of Pim inhibitors. These results suggest that the Pim protein kinase can regulate stromal cell biology to modulate epithelial

  1. CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Hieu T Nim

    Full Text Available The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP, an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1 relevant to cardiac literature, and (2 differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10 are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

  2. CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

    Science.gov (United States)

    Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

    2015-01-01

    The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

  3. Conceptualising the lack of health insurance coverage.

    Science.gov (United States)

    Davis, J B

    2000-01-01

    This paper examines the lack of health insurance coverage in the US as a public policy issue. It first compares the problem of health insurance coverage to the problem of unemployment to show that in terms of the numbers of individuals affected lack of health insurance is a problem comparable in importance to the problem of unemployment. Secondly, the paper discusses the methodology involved in measuring health insurance coverage, and argues that the current method of estimation of the uninsured underestimates the extent that individuals go without health insurance. Third, the paper briefly introduces Amartya Sen's functioning and capabilities framework to suggest a way of representing the extent to which individuals are uninsured. Fourth, the paper sketches a means of operationalizing the Sen representation of the uninsured in terms of the disability-adjusted life year (DALY) measure.

  4. Laura: Soybean variety lacking Kunitz trypsin inhibitor

    Directory of Open Access Journals (Sweden)

    Srebrić Mirjana

    2010-01-01

    Full Text Available Grain of conventional soybean varieties requires heat processing to break down trypsin inhibitor's activity before using as food or animal feed. At the same time, protein denaturation and other qualitative changes occur in soybean grain, especially if the temperature of heating is not controlled. Two types of trypsin inhibitor were found in soybean grain the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor. Mature grain of soybean Laura is lacking Kunitz trypsin inhibitor. Grain yield of variety Laura is equal to high yielding varieties from the maturity group I, where it belongs. Lacking of Kunitz-trypsin inhibitor makes soybean grain suitable for direct feeding in adult non ruminant animals without previous thermal processing. Grain of variety Laura can be processed for a shorter period of time than conventional soybeans. This way we save energy, and preserve valuable nutritional composition of soybean grain, which is of interest in industrial processing.

  5. Fibroblast Growth Factor Signaling in Metabolic Regulation.

    Science.gov (United States)

    Nies, Vera J M; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T; Atkins, Annette R; Evans, Ronald M; Jonker, Johan W; Downes, Michael Robert

    2015-01-01

    The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.

  6. Senescent phenotypes of skin fibroblasts from patients with Tangier disease

    International Nuclear Information System (INIS)

    Matsuura, Fumihiko; Hirano, Ken-ichi; Ikegami, Chiaki; Sandoval, Jose C.; Oku, Hiroyuki; Yuasa-Kawase, Miyako; Tsubakio-Yamamoto, Kazumi; Koseki, Masahiro; Masuda, Daisaku; Tsujii, Ken-ichi; Ishigami, Masato; Nishida, Makoto; Shimomura, Iichiro; Hori, Masatsugu; Yamashita, Shizuya

    2007-01-01

    Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) ∼10 compared with control cells. TD cells practically ceased proliferation at PDL ∼30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated β-galactosidase (SA-β-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients

  7. [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

    Science.gov (United States)

    Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

    2017-01-01

    Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

  8. 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

  9. Phenotype change and migration of adventitial fibroblasts during postangioplasty

    International Nuclear Information System (INIS)

    Wang Yongli; Zhang Jiaxing; He Nengshu; Si Tongguo; Fan Hailun; Ge Xihong; Xu Rui

    2006-01-01

    Objective: To verify fibroblasts translocation from adventitia into neointima by labeling adventitia cells with bromodeoxyuridine (BrDU) after angioplasty, and to explore the relationship of adventitial fibroblast with restenosis. Methods: Vascular restenosis model was created by injured intima of common carotid artery (CCA) of mouse with guide wire, adventitial fibroblasts were labeled with BrDU, and dynamic distribution of myofibroblasts in adventitia, media and neoitima was observed at different times (3 d, 7 d, 14 d and 28 d) by means of single/double-label immunohistochemistry, light microscope, electronic microscope and image analysis system. Results: 1.Immunohistochemistry: More adventitial fibroblasts combined with BrDU could be found in adventitia on the 3rd day of postangioplasty, and the number of this kind of cells reached the peak on 7th day, and at the same time fibroblasts changed their phenotypes and became myofibroblasts, which produced α-actin and extracellular matrix (ECM). On 14th day, the number of the positive cells decreased in adventitia, increased in media and neointima associated with intima thickening; on 28th day, while the number of fibroblasts labeled by BrDU returned to the basic-line in adventitia, media and intima, nevertheless, intima thickening and vascular stenosis and intimal ELM precipitation were still present. There were significant differences in the number of fibroblasts labeled with BrDU located in three layers of artery (P<0.05). 2. Electronic microscope: After angioplasty, the plasm of fibroblasts became rich, mitochondrious and increase of Golgi apparatus; and the amount of rough endoplasmic reticulums rose with more secretory granules, together with a great amount of collagen synthesized forming the microfilaments; on days of 7th and 14th, the wide pseudopodia of myofibroblasts could be found extending into the windows on the external elastic lamina (ELL) and the internal elastic lamina (ILL); and showing the tendency

  10. Accidents in radiotherapy: Lack of quality assurance?

    International Nuclear Information System (INIS)

    Novotny, J.

    1997-01-01

    About 150 radiological accidents, involving more than 3000 patients with adverse effects, 15 patient's fatalities and about 5000 staff and public exposures have been collected and analysed. Out of 67 analysed accidents in external beam therapy 22% has been caused by wrong calculation of the exposure time or monitor units, 13% by inadequate review of patient's chart, 12% by mistakes in the anatomical area to be treated. The remaining 35% can be attributed to 17 different causes. The most common mistakes in brachytherapy were wrong activities of sources used for treatment (20%), inadequate procedures for placement of sources applicators (14%), mistakes in calculating the treatment time (12%), etc. The direct and contributing causes of radiological accidents have been deduced from each event, when it was possible and categorized into 9 categories: mistakes in procedures (30%), professional mistakes (17%), communication mistakes (15%), lack of training (8.5%), interpretation mistakes (7%), lack of supervision (6%), mistakes in judgement (6%), hardware failures (5%), software and other mistakes (5.5%). Three types of direct and contributing causes responsible for almost 62% of all accidents are directly connected to the quality assurance of treatment. The lessons learnt from the accidents are related to frequencies of direct and contributing factors and show that most of the accident are caused by lack, non-application of quality assurance (QA) procedures or by underestimating of QA procedures. The international system for collection of accidents and dissemination of lessons learnt from the different accidents, proposed by IAEA, can contribute to better practice in many radiotherapy departments. Most of the accidents could have been avoided, had a comprehensive QA programme been established and properly applied in all radiotherapy departments, whatever the size. (author)

  11. Why does Colombia lack agricultural commodity futures?

    Directory of Open Access Journals (Sweden)

    Pablo Moreno-Alemay

    2015-11-01

    Full Text Available This article explores the reasons why futures contracts are not traded as an alternative to price hedging for agricultural goods in Colombia. Based on surveys, interviews and statistical analysis, this study identified that conceptual gaps in contract negotiation, lack of consensus in the agricultural sector regarding the use of financial mechanisms and the sector’s infrequent contact with Colombia’s financial institutions, are the main reasons why a futures contracts market has not emerged.

  12. Fibroblast growth factor signaling in metabolic regulation

    Directory of Open Access Journals (Sweden)

    Vera eNies

    2016-01-01

    Full Text Available The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases, and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed.In this review we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease, and to provide starting points for the development of FGF-based therapies against metabolic conditions.

  13. Physiological Actions of Fibroblast Growth Factor-23

    Directory of Open Access Journals (Sweden)

    Reinhold G. Erben

    2018-05-01

    Full Text Available Fibroblast growth factor-23 (FGF23 is a bone-derived hormone suppressing phosphate reabsorption and vitamin D hormone synthesis in the kidney. At physiological concentrations of the hormone, the endocrine actions of FGF23 in the kidney are αKlotho-dependent, because high-affinity binding of FGF23 to FGF receptors requires the presence of the co-receptor αKlotho on target cells. It is well established that excessive concentrations of intact FGF23 in the blood lead to phosphate wasting in patients with normal kidney function. Based on the importance of diseases associated with gain of FGF23 function such as phosphate-wasting diseases and chronic kidney disease, a large body of literature has focused on the pathophysiological consequences of FGF23 excess. Less emphasis has been put on the role of FGF23 in normal physiology. Nevertheless, during recent years, lessons we have learned from loss-of-function models have shown that besides the paramount physiological roles of FGF23 in the control of 1α-hydroxylase expression and of apical membrane expression of sodium-phosphate co-transporters in proximal renal tubules, FGF23 also is an important stimulator of calcium and sodium reabsorption in distal renal tubules. In addition, there is an emerging role of FGF23 as an auto-/paracrine regulator of alkaline phosphatase expression and mineralization in bone. In contrast to the renal actions of FGF23, the FGF23-mediated suppression of alkaline phosphatase in bone is αKlotho-independent. Moreover, FGF23 may be a physiological suppressor of differentiation of hematopoietic stem cells into the erythroid lineage in the bone microenvironment. At present, there is little evidence for a physiological role of FGF23 in organs other than kidney and bone. The purpose of this mini-review is to highlight the current knowledge about the complex physiological functions of FGF23.

  14. Fatty acid effects on fibroblast cholesterol synthesis

    International Nuclear Information System (INIS)

    Shireman, R.B.; Muth, J.; Lopez, C.

    1987-01-01

    Two cell lines of normal (CRL 1475, GM5565) and of familial hypercholesterolemia (FH) (CM 486,488) fibroblasts were preincubated with medium containing the growth factor ITS, 2.5 mg/ml fatty acid-free BSA, or 35.2 μmol/ml of these fatty acids complexed with 2.5 mg BSA/ml: stearic (18:0), caprylic (8:0), oleic (18:1;9), linoleic (18:2;9,12), linolenic (18:3;9,12,15), docosahexaenoic (22:6;4,7,10,13,16,19)(DHA) or eicosapentaenoic (20:5;5,8,11,14,17)(EPA). After 20 h, cells were incubated for 2 h with 0.2 μCi [ 14 C]acetate/ml. Cells were hydrolyzed; an aliquot was quantitated for radioactivity and protein. After saponification and extraction with hexane, radioactivity in the aqueous and organic phases was determined. The FH cells always incorporated 30-90% more acetate/mg protein than normal cells but the pattern of the fatty acid effects was similar in both types. When the values were normalized to 1 for the BSA-only group, cells with ITS had the greatest [ 14 C]acetate incorporation (1.45) followed by the caprylic group (1.14). Cells incubated with 18:3, 20:6 or 22:6 incorporated about the same amount as BSA-only. Those preincubated with 18:2, 18:1, 18:0 showed the least acetate incorporation (0.87, 0.59 and 0.52, respectively). The percentage of total 14 C counts which extracted into hexane was much greater in FH cells; however, these values varied with the fatty acid, e.g., 1.31(18:0) and 0.84(8:0) relative to 1

  15. Fibroblasts and myofibroblasts in wound healing

    Directory of Open Access Journals (Sweden)

    Darby IA

    2014-11-01

    Full Text Available Ian A Darby,1 Betty Laverdet,2 Frédéric Bonté3, Alexis Desmoulière2 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia; 2Department of Physiology and EA 6309, FR 3503, Faculties of Medicine and Pharmacy, University of Limoges, Limoges, France; 3LVMH Recherche, Saint Jean de Braye, France Abstract: (Myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myofibroblasts are embedded in a sophisticated extracellular matrix (ECM that they secrete, and a complex and interactive dialogue exists between (myofibroblasts and their microenvironment. In addition to the secretion of the ECM, (myofibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myofibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myofibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis, or during aging, this dialogue between the (myofibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myofibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment. Keywords: myofibroblast, fibroblast, α-smooth muscle actin

  16. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  17. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  18. Reduced alcohol consumption in mice lacking preprodynorphin.

    Science.gov (United States)

    Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

    2006-10-01

    Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

  19. Increased fibroblast functionality on CNN2-loaded titania nanotubes

    Directory of Open Access Journals (Sweden)

    Wei HB

    2012-02-01

    Full Text Available Hongbo Wei*, Shuyi Wu*, Zhihong Feng, Wei Zhou, Yan Dong, Guofeng Wu, Shizhu Bai, Yimin Zhao Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China *These authors contributed equally to this workAbstract: Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.Keywords: anodization, titania nanotubes, adhesion, connective

  20. Binding, uptake, and release of nicotine by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Hanes, P.J.; Schuster, G.S.; Lubas, S.

    1991-01-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4 degree C using a mixture of 3 H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3 H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37 degree C after treating cells with 3 H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours

  1. PROPOSED CARDIAC STEM CELLS DERIVED FROM “CARDIOSPHERES” LACK CARDIOMYOGENIC POTENTIAL

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline

       Recent studies have reported that clinical relevant numbers of cardiac stem cells (CSCs) with cardiomyogenic potential can be obtained from small heart tissue biopsies, by an intrinsic ability of CSCs to form beating cardiospheres (CSs) during ex vivo culture. Such data have provided optimism...... that injuried heart tissue may be repaired by stem cell therapy using autologous CS derived cells, and pre-clinical studies have already been described in literature.    Herein, we established CSs from neonatal rats, and by immunofluorescence, qRT-PCR, and microscopic examination we demonstrated...... to form CSs by themselves. Phenotypically, CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential.    Our data imply that at least the murine cardiosphere model seems unsuitable for enrichment of cardiac stem cells with cardiomyogenic...

  2. Lack of consensus in social systems

    Science.gov (United States)

    Benczik, I. J.; Benczik, S. Z.; Schmittmann, B.; Zia, R. K. P.

    2008-05-01

    We propose an exactly solvable model for the dynamics of voters in a two-party system. The opinion formation process is modeled on a random network of agents. The dynamical nature of interpersonal relations is also reflected in the model, as the connections in the network evolve with the dynamics of the voters. In the infinite time limit, an exact solution predicts the emergence of consensus, for arbitrary initial conditions. However, before consensus is reached, two different metastable states can persist for exponentially long times. One state reflects a perfect balancing of opinions, the other reflects a completely static situation. An estimate of the associated lifetimes suggests that lack of consensus is typical for large systems.

  3. Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    François Huaux

    Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

  4. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

    Science.gov (United States)

    Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

    2017-01-01

    Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing. PMID:28773152

  5. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  6. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

    Directory of Open Access Journals (Sweden)

    Rebeca Illescas-Montes

    2017-07-01

    Full Text Available Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2 using different transmission modes (continuous or pulsed. The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing.

  7. DNA damage in cultured human skin fibroblasts exposed to excimer laser radiation

    Energy Technology Data Exchange (ETDEWEB)

    Rimoldi, D.; Miller, A.C.; Freeman, S.E.; Samid, D. (Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD (USA))

    1991-06-01

    Ultraviolet excimer lasers are being considered for use in a variety of refractive and therapeutic procedures, the long-term biologic consequences of which are unknown. The effect of sublethal doses of 193-nm laser radiation on cellular DNA was examined in cultured human skin fibroblasts. In contrast to 248 nm, treatments with the 193-nm laser radiation below 70 J/m2 did not cause significant pyrimidine dimer formation in the skin cells. This was indicated by the lack of excision repair activities (unscheduled DNA synthesis assay), and further demonstrated by direct analysis of pyrimidine dimers in DNA from irradiated cells. However, a low level of unscheduled DNA synthesis could be detected following irradiation at 193 nm with 70 J/m2. Both the 193-nm and 248-nm radiation were able to induce chromosomal aberrations, as indicated by a micronucleus assay. A dose-dependent increase in micronuclei frequency was observed 48 and 72 h after laser irradiation. These results indicate that exposure of actively replicating human skin fibroblasts to sublethal doses of either 193- or 248-nm laser radiation can result in genotoxicity.

  8. Engraftment potential of dermal fibroblasts following in vivo myogenic conversion in immunocompetent dystrophic skeletal muscle

    Directory of Open Access Journals (Sweden)

    Lindsey A Muir

    2014-01-01

    Full Text Available Autologous dermal fibroblasts (dFbs are promising candidates for enhancing muscle regeneration in Duchenne muscular dystrophy (DMD due to their ease of isolation, immunological compatibility, and greater proliferative potential than DMD satellite cells. We previously showed that mouse fibroblasts, after MyoD-mediated myogenic reprogramming in vivo, engraft in skeletal muscle and supply dystrophin. Assessing the therapeutic utility of this system requires optimization of conversion and transplantation conditions and quantitation of engraftment so that these parameters can be correlated with possible functional improvements. Here, we derived dFbs from transgenic mice carrying mini-dystrophin, transduced them by lentivirus carrying tamoxifen-inducible MyoD, and characterized their myogenic and engraftment potential. After cell transplantation into the muscles of immunocompetent dystrophic mdx4cv mice, tamoxifen treatment drove myogenic conversion and fusion into myofibers that expressed high levels of mini-dystrophin. Injecting 50,000 cells/µl (1 × 106 total cells resulted in a peak of ∼600 mini-dystrophin positive myofibers in tibialis anterior muscle single cross-sections. However, extensor digitorum longus muscles with up to 30% regional engraftment showed no functional improvements; similar limitations were obtained with whole muscle mononuclear cells. Despite the current lack of physiological improvement, this study suggests a viable initial strategy for using a patient-accessible dermal cell population to enhance skeletal muscle regeneration in DMD.

  9. Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts.

    Science.gov (United States)

    Yang, Zhenzhen; Vajta, Gábor; Xu, Ying; Luan, Jing; Lin, Mufei; Liu, Cong; Tian, Jianing; Dou, Hongwei; Li, Yong; Liu, Tianbin; Zhang, Yijie; Li, Lin; Yang, Wenxian; Bolund, Lars; Yang, Huanming; Du, Yutao

    2016-08-01

    Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.

  10. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  11. Biosynthesis of collagen by fibroblasts kept in culture

    International Nuclear Information System (INIS)

    Machado-Santelli, G.M.

    1978-01-01

    The sinthesis of collagen is studied in fibroblasts of different origins with the purpose of obtaining an appropriate system for the study of its biosynthesis and processing. The percentage of collagen synthesis vary according to the fibroblast origin. Experiences are performed with fibroblasts kept in culture from: chicken - and guinea pig embryos, carragheenin - induced granulomas in adult guinea pig and from human skin. The collagen pattern synthesized after acetic acid - or saline extractions in the presence of inhibitors is also determined. This pattern is then assayed by poliacrilamide - 5% - SDS gel electrophoresis accompanied by fluorography. The importance of the cell culture system in the elucidation of collagen biosynthesis is pointed out. (M.A.) [pt

  12. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    Science.gov (United States)

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

  13. 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.

  14. 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.

  15. The Possible Pre- and Post-UVA Radiation Protective Effect of Amaranth Oil on Human Skin Fibroblast Cells.

    Science.gov (United States)

    Wolosik, Katarzyna; Zareba, Ilona; Surazynski, Arkadiusz; Markowska, Agnieszka

    2017-07-01

    The health effects of Amaranth Oil (AO) are attributed to its specific chemical composition. That makes it an outstanding natural product for the prevention and treatment of ultraviolet (UV) irradiation-related pathologies such as sunburn, photoaging, photoimmunosuppression, and photocarcinogenesis. Most of the studies are taken on animal model, and there is a lack of research on the endogenous effect of AO on fibroblast level, where UVA takes it harmful place. The aim of this study was evaluation if AO can protect or abolish UVA exposure effect on human skin fibroblast. The 0.1% AO, 0.25% AO, and 0.5% AO concentration and irradiation for 15 min under UVA-emitting lamp were studied in various condition. In all experiments, the mean values for six assays ± standard deviations were calculated. Pretreatment with various concentrations of AO was tested. The highest concentration of AO where cell survival was observed was 0.5%. Cytotoxicity assays provided evidence for pre- and post-UVA protective effect of 0.1% AO among three tested concentrations. The results also provide evidence that UVA has inhibitory effect on collagen biosynthesis in confluent skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect comparing to other used AO concentration. The assessment results on DNA biosynthesis show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added. AO gives pre- and post-UVA protection in low concentration. This provides the evidence for using it not as a main protective factor against UV but as one of the combined components in cosmetic formulation. The recommended Amaranth Oil (AO) concentration in cosmetic formulation is between 0.1 and 5%Pretreatment with various concentrations of AO suggests to use the highest 0.5% concentration of AO in human skin fibroblast culturesThe 0.1% of AO in fibroblast cultures, protects and abolishes effect of ultraviolet A (UVA) exposureUVA has inhibitory effect on collagen biosynthesis in

  16. Multifaced Roles of the αvβ3 Integrin in Ehlers–Danlos and Arterial Tortuosity Syndromes’ Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Nicoletta Zoppi

    2018-03-01

    Full Text Available The αvβ3 integrin, an endothelial cells’ receptor-binding fibronectin (FN in the extracellular matrix (ECM of blood vessels, regulates ECM remodeling during migration, invasion, angiogenesis, wound healing and inflammation, and is also involved in the epithelial mesenchymal transition. In vitro-grown human control fibroblasts organize a fibrillar network of FN, which is preferentially bound on the entire cell surface to its canonical α5β1 integrin receptor, whereas the αvβ3 integrin is present only in rare patches in focal contacts. We report on the preferential recruitment of the αvβ3 integrin, due to the lack of FN–ECM and its canonical integrin receptor, in dermal fibroblasts from Ehlers–Danlos syndromes (EDS and arterial tortuosity syndrome (ATS, which are rare multisystem connective tissue disorders. We review our previous findings that unraveled different biological mechanisms elicited by the αvβ3 integrin in fibroblasts derived from patients affected with classical (cEDS, vascular (vEDS, hypermobile EDS (hEDS, hypermobility spectrum disorders (HSD, and ATS. In cEDS and vEDS, respectively, due to defective type V and type III collagens, αvβ3 rescues patients’ fibroblasts from anoikis through a paxillin-p60Src-mediated cross-talk with the EGF receptor. In hEDS and HSD, without a defined molecular basis, the αvβ3 integrin transduces to the ILK-Snail1-axis inducing a fibroblast-to-myofibroblast-transition. In ATS cells, the deficiency of the dehydroascorbic acid transporter GLUT10 leads to redox imbalance, ECM disarray together with the activation of a non-canonical αvβ3 integrin-TGFBRII signaling, involving p125FAK/p60Src/p38MAPK. The characterization of these different biological functions triggered by αvβ3 provides insights into the multifaced nature of this integrin, at least in cultured dermal fibroblasts, offering future perspectives for research in this field.

  17. Agminated Fibroblastic Conective Tissue Nevus: A New Clinical Presentation.

    Science.gov (United States)

    Downey, Camila; Requena, Luis; Bagué, Silvia; Sánchez Martínez, Miquel Ángel; Lloreta, Josep; Baselga, Eulalia

    2016-07-01

    Connective tissue nevi are benign hamartomatous lesions in which one or several of the components of the dermis (collagen, elastin, glicosaminoglycans) show predominance or depletion. Recently, de Feraudy et al broadened the spectrum of connective tissue nevus, describing fibroblastic connective tissue nevus (FCTN), which is characterized by proliferation of CD34(+) cells of fibroblastic and myofibroblastic lineage. Only solitary papules and nodules have been described. We present the first case of FCTN with multiple agminated lesions on the leg of an infant and the difficulties encountered in the differential diagnosis with dermatofibrosarcoma protuberans. © 2016 Wiley Periodicals, Inc.

  18. Chloride transport in human fibroblasts is activated by hypotonic shock

    Energy Technology Data Exchange (ETDEWEB)

    Rugolo, M.; Mastocola, T.; Flamigni, A.; Lenaz, G. (Universita' di Bologna (Italy))

    1989-05-15

    Incubation of human skin fibroblasts in hypotonic media induced the activation of {sup 36}Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of {sup 36}Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also {sup 36}Cl- influx was enhanced by hypotonic medium.

  19. Mactosylceramide Prevents Glial Cell Overgrowth by Inhibiting Insulin and Fibroblast Growth Factor Receptor Signaling

    DEFF Research Database (Denmark)

    Gerdøe-Kristensen, Stine; Lund, Viktor K; Wandall, Hans H

    2017-01-01

    , in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what...... of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK...... hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia. This article is protected by copyright. All rights reserved....

  20. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    International Nuclear Information System (INIS)

    Kakudo, Natsuko; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-01-01

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration

  1. Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

    Directory of Open Access Journals (Sweden)

    Chiquita Prahasanti

    2018-04-01

    Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

  2. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    OpenAIRE

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine r...

  3. Lack of RNase L attenuates macrophage functions.

    Directory of Open Access Journals (Sweden)

    Xin Yi

    Full Text Available Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α, interleukin (IL-1β, IL-6, and cyclooxygenase-2 (Cox-2 by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.Bone marrow-derived macrophages (BMMs were generated from RNase L(+/+and (-/- mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2. Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

  4. Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema

    NARCIS (Netherlands)

    Noordhoek, JA; Postma, DS; Chong, LL; Vos, JTWM; Kauffman, HF; Timens, W; van Straaten, JFM

    2003-01-01

    To characterize the possible role of a dysregulated proliferative capacity of pulmonary fibroblasts in insufficient tissue repair in lungs from patients with pulmonary emphysema, the authors undertook in vitro proliferative studies with pulmonary fibroblasts obtained from lung tissue of patients

  5. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

    NARCIS (Netherlands)

    Scheres, N; Laine, M L; de Vries, T J; Everts, V; van Winkelhoff, A J

    BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can

  6. Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

    DEFF Research Database (Denmark)

    Dabelsteen, S; Wandall, H H; Grøn, B

    1997-01-01

    Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m......RNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal...

  7. Lack of efficacy of ergocalciferol repletion

    Directory of Open Access Journals (Sweden)

    Thomas Wasser

    2012-04-01

    Full Text Available Introduction: Vitamin D has become an area of intensive scrutiny, both in medical and lay literature. However, there are limited data to suggest proper repletion regimens for those patients who have hypovitaminosis D. Consequently, various methods are used in clinical practice. The aim of this study was to assess the efficacy of various treatment strategies for hypovitaminosis D in an ambulatory internal medicine practice. Methods: A retrospective chart review between October 2005 and June 2010 of a suburban internal medicine practice was performed via query of the electronic medical record (Centricity, General Electric Healthcare, UK. Patients with a 25-hydroxyvitamin D concentration less than 32 mg/dl were identified and treated. Treatment success was defined as 25-hydroxyvitamin D concentrations greater than 32 mg/dl. Statistical analysis to assess changes in vitamin D level controlling for season, comorbidities, and demographics were used. Results: A total of 607 treatment episodes were identified, with 395 excluded due to lack of follow-up vitamin D level within 16 weeks, no treatment documented, topical treatment, doxercalciferol treatment, or non-compliance. Of the remaining patients, there were 212 treatment instances on 178 patients. Ergocalciferol 50,000 international units (IU was used most frequently (71.4% of the time.. A higher initial vitamin D level was positively associated with treatment success (adjusted odds ratio = 1.11, p=0.002. Increased doses of ergocalciferol increased the likelihood of treatment success (p=0.0011. Seasonal variation was related to posttreatment 25-hydroxyvitamin D concentration as was body mass index (BMI (p=0.003 and p=0.044. Conclusion: Pretreatment levels of 25-hydroxyvitamin D, BMI, season, and vitamin D dose are predictors of successful hypovitaminosis D treatment. Our data suggest that patients with initial 25-hydroxyvitamin D concentrations of <20 should be treated with a higher total dose of

  8. 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...

  9. Constitutive Reprogramming of Fibroblast Mitochondrial Metabolism in Pulmonary Hypertension

    Czech Academy of Sciences Publication Activity Database

    Plecitá-Hlavatá, Lydie; Tauber, Jan; Li, M.; Zhang, H.; Flockton, A. R.; Pullamsetti, S. S.; Chelladurai, P.; D'Alessandro, A.; El Kasmi, K. C.; Ježek, Petr; Stenmark, K. R.

    2016-01-01

    Roč. 55, č. 1 (2016), s. 47-57 ISSN 1044-1549 R&D Projects: GA MŠk(CZ) LH11055; GA MŠk(CZ) LH15071 Institutional support: RVO:67985823 Keywords : mitochondria * complex I * oxidative metabolism * pulmonary hypertension * adventitial fibroblasts Subject RIV: ED - Physiology Impact factor: 4.100, year: 2016

  10. Fibroblast Growth Factor-2 Alters the Nature of Extinction

    Science.gov (United States)

    Graham, Bronwyn M.; Richardson, Rick

    2011-01-01

    These experiments examined the effects of the NMDA-receptor (NMDAr) antagonist MK801 on reacquisition and re-extinction of a conditioned fear that had been previously extinguished before injection of fibroblast growth factor-2 (FGF2) or vehicle. Recent findings have shown that relearning and re-extinction, unlike initial learning and extinction,…

  11. Arachidonic acid metabolism in fibroblasts derived from canine myocardium

    International Nuclear Information System (INIS)

    Weber, D.R.; Prescott, S.M.

    1986-01-01

    Canine fibroblasts from normal or healing infarcted myocardium were grown in culture. The cells were morphologically indistinguishable, but the doubling time of cells from healing myocardium was 39.6 +/- 3.5 hr whereas that of normals was 24 +/- 3.7 (n=5, p 3 H]arachidonate (AA) into phospholipids. Calcium ionophore A23187 (10 μM) caused release and metabolism of [ 3 H] AA. A23187 or AA (10μM) induced production of 6-keto PGF1α, PGE2, and a hydroxy metabolite of AA. RIA of 6-keto PGF1α showed that subconfluent cells from healing myocardium produced 1202 +/- 354 pg/mg protein whereas that of normals was 551 +/- 222 (n=7, p 3 H]AA released but did not metabolize [ 3 H]AA. In coincubations, fibroblasts incorporated myocyte-derived AA. Subsequent stimulation of the fibroblasts with A23187 induced the synthesis of 6-keto PGF1α, PGE2 and a hydroxy metabolite. The fibroblast content of healing myocardium was 35-1000 times that of normal tissue (n=7). Thus even a moderate change in AA metabolism, amplified by the AA released from deteriorating myocytes, may be a significant physiologic or pathologic event

  12. Transfer of fibroblast sheets cultured on thermoresponsive dishes with membranes.

    Science.gov (United States)

    Kawecki, Marek; Kraut, Małgorzata; Klama-Baryła, Agnieszka; Łabuś, Wojciech; Kitala, Diana; Nowak, Mariusz; Glik, Justyna; Sieroń, Aleksander L; Utrata-Wesołek, Alicja; Trzebicka, Barbara; Dworak, Andrzej; Szweda, Dawid

    2016-06-01

    In cell or tissue engineering, it is essential to develop a support for cell-to-cell adhesion, which leads to the generation of cell sheets connected by extracellular matrix. Such supports must be hydrophobic and should result in a detachable cell sheet. A thermoresponsive support that enables the cultured cell sheet to detach using only a change in temperature could be an interesting alternative in regenerative medicine. The aim of this study was to evaluate plates covered with thermoresponsive polymers as supports for the formation of fibroblast sheets and to develop a damage-free procedure for cell sheet transfer with the use of membranes as transfer tools. Human skin fibroblasts were seeded on supports coated with a thermoresponsive polymer: commercial UpCell™ dishes (NUNC™) coated with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and dishes coated with thermoresponsive poly(tri(ethylene glycol) monoethyl ether methacrylate) (P(TEGMA-EE)). Confluent fibroblast sheets were effectively cultured and harvested from both commercial PNIPAM-coated dishes and laboratory P(TEGMA-EE)-coated dishes. To transfer a detached cell sheet, two membranes, Immobilon-P(®) and SUPRATHEL(®), were examined. The use of SUPRATHEL for relocating the cell sheets opens a new possibility for the clinical treatment of wounds. This study established the background for implementing thermoresponsive supports for transplanting in vitro cultured fibroblasts.

  13. Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

    Science.gov (United States)

    Lee, Yoon; Ahn, Jin-Soo; Yi, Young-Ah; Chung, Shin-Hye; Yoo, Yeon-Jee; Ju, Sung-Won; Hwang, Ji-Yun; Seo, Deog-Gyu

    2015-02-01

    The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

  14. fibroblast growth factor, MTDH/Astrocyte elevated gene-1

    African Journals Online (AJOL)

    2012-12-05

    Dec 5, 2012 ... Expression of basic FGF, MTDH/AEG-1, APC, matrix metalloproteinase 9, and COX-2 markers in prostate carcinomas many genetic and epigenetic alterations have been detected in human PC.[1]. Basic fibroblast growth factor (bFGF), also known as. FGF2 is a member of the FGF family, a group of more.

  15. Involvement of the mitochondrial compartment in human NCL fibroblasts

    International Nuclear Information System (INIS)

    Pezzini, Francesco; Gismondi, Floriana; Tessa, Alessandra; Tonin, Paola; Carrozzo, Rosalba; Mole, Sara E.; Santorelli, Filippo M.; Simonati, Alessandro

    2011-01-01

    Highlights: ► Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. ► Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. ► Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  16. Primary skin fibroblasts as a model of Parkinson's disease

    NARCIS (Netherlands)

    Auburger, G.; Klinkenberg, M.; Droste, J.A.H.; Marcus, K.; Morales-Gordo, B.; Kunz, W.S.; Brandt, U.; Broccoli, V.; Reichmann, H.; Gispert, S.; Jendrach, M.

    2012-01-01

    Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts

  17. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    International Nuclear Information System (INIS)

    Jessup, W.; Dean, R.T.

    1983-01-01

    Normal rates of pinocytosis of [ 3 H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  18. DETACHMENT OF HUMAN FIBROBLASTS FROM FEP-TEFLON SURFACES

    NARCIS (Netherlands)

    VANKOOTEN, TG; SCHAKENRAAD, JM; VANDERMEI, HC; BUSSCHER, HJ

    1991-01-01

    In this study a comparison is made between the detachment behavior of human fibroblasts adhered to hydrophobic FEP-Teflon (water contact angle 109 degrees) and to hydrophilic glass (water contact angle smaller than 15 degrees) during exposure to a laminar, incrementally loaded flow. Detachment from

  19. Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

    Science.gov (United States)

    Gupta, Manoj K; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F; Windmueller, Rebecca; Wagers, Amy J; Kulkarni, Rohit N

    2015-10-01

    The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. ©AlphaMed Press.

  20. Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies

    NARCIS (Netherlands)

    Koster, Janet; Waterham, Hans R.

    2017-01-01

    Functional studies with primary human skin fibroblasts from patients with a peroxisomal disorder often require efficient transfection with plasmids to correct the genetic defect or to express heterologous reporter proteins. Here, we describe a protocol we commonly use for efficient nonviral

  1. Scleral fibroblast response to experimental glaucoma in mice

    Science.gov (United States)

    Tezel, Gülgün; Cone-Kimball, Elizabeth; Steinhart, Matthew R.; Jefferys, Joan; Pease, Mary E.; Quigley, Harry A.

    2016-01-01

    Purpose To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. Methods Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using 16O/18O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4’,6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. Results Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. Conclusions Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition. PMID:26900327

  2. Instability restricts signaling of multiple fibroblast growth factors

    Czech Academy of Sciences Publication Activity Database

    Buchtová, Marcela; Chaloupková, R.; Zakrzewska, M.; Veselá, I.; Celá, Petra; Barathová, J.; Gudernová, I.; Zajíčková, R.; Trantírek, L.; Martin, J.; Kostas, M.; Otlewski, J.; Damborský, J.; Kozubík, Alois; Wiedlocha, A.; Krejčí, P.

    2015-01-01

    Roč. 72, č. 12 (2015), s. 2445-2459 ISSN 1420-682X R&D Projects: GA ČR(CZ) GA14-31540S; GA ČR GBP302/12/G157 Institutional support: RVO:67985904 ; RVO:68081707 Keywords : fibroblast growth factor * FGF * unstable Subject RIV: EA - Cell Biology Impact factor: 5.694, year: 2015

  3. Entrainment of Spontaneously Hypertensive Rat Fibroblasts by Temperature Cycles

    Science.gov (United States)

    Sládek, Martin; Sumová, Alena

    2013-01-01

    The functional state of the circadian system of spontaneously hypertensive rats (SHR) differs in several characteristics from the functional state of normotensive Wistar rats. Some of these changes might be due to the compromised ability of the central pacemaker to entrain the peripheral clocks. Daily body temperature cycles represent one of the important cues responsible for the integrity of the circadian system, because these cycles are driven by the central pacemaker and are able to entrain the peripheral clocks. This study tested the hypothesis that the aberrant peripheral clock entrainment of SHR results from a compromised peripheral clock sensitivity to the daily temperature cycle resetting. Using cultured Wistar rat and SHR fibroblasts transfected with the circadian luminescence reporter Bmal1-dLuc, we demonstrated that two consecutive square-wave temperature cycles with amplitudes of 2.5°C are necessary and sufficient to restart the dampened oscillations and entrain the circadian clocks in both Wistar rat and SHR fibroblasts. We also generated a phase response curve to temperature cycles for fibroblasts of both rat strains. Although some of the data suggested a slight resistance of SHR fibroblasts to temperature entrainment, we concluded that the overall effect it too weak to be responsible for the differences between the SHR and Wistar in vivo circadian phenotype. PMID:24116198

  4. Chenodeoxycholic acid stimulated fibroblast growth factor 19 response

    DEFF Research Database (Denmark)

    Borup, C; Wildt, S; Rumessen, J J

    2017-01-01

    BACKGROUND: Bile acid diarrhoea is underdiagnosed and better diagnostic tests are needed. Fasting serum fibroblast growth factor-19 (FGF19) has insufficient diagnostic value, but this may be improved by stimulation. AIM: To explore if an impaired FGF19 response identifies primary bile acid...

  5. Anti-fibrotic effects of theophylline on lung fibroblasts

    International Nuclear Information System (INIS)

    Yano, Yukihiro; Yoshida, Mitsuhiro; Hoshino, Shigenori; Inoue, Koji; Kida, Hiroshi; Yanagita, Masahiko; Takimoto, Takayuki; Hirata, Haruhiko; Kijima, Takashi; Kumagai, Toru; Osaki, Tadashi; Tachibana, Isao; Kawase, Ichiro

    2006-01-01

    Theophylline has been used in the management of bronchial asthma and chronic obstructive pulmonary disease for over 50 years. It has not only a bronchodilating effect, but also an anti-inflammatory one conducive to the inhibition of airway remodeling, including subepithelial fibrosis. To date however, whether theophylline has a direct inhibitory effect on airway fibrosis has not been established. To clarify this question, we examined whether theophylline affected the function of lung fibroblasts. Theophylline suppressed TGF-β-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation stimulated by FBS and TGF-β-induced α-SMA protein. A cAMP analog also inhibited TGF-β-induced COL1 mRNA expression in lung fibroblasts. A PKA inhibitor reduced the inhibitory effect of theophylline on TGF-β-induced COL1 mRNA expression. These results indicate that theophylline exerts anti-fibrotic effects, at least partly, through the cAMP-PKA pathway

  6. The effects of acoustic vibration on fibroblast cell migration.

    Science.gov (United States)

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

    Directory of Open Access Journals (Sweden)

    Xuan Wang

    2016-05-01

    Full Text Available AIM: To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon’s capsule, both in vitro and in vivo. METHODS: Human primary Tenon’s capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2, tissue inhibitor of metalloproteinase (TIMP-1,2 and proliferating cell nuclear antigen (PCNA. Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d. A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson’s trichrome to compare the neovascularization in the subconjunctiva area. RESULTS: In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson’s trichrome observation. CONCLUSION: By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

  8. 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

  9. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-01-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with 125 I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture

  10. Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

    Directory of Open Access Journals (Sweden)

    Kristin Dawson

    Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

  11. 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.

  12. The impact of vascular endothelial growth factor and basic fibroblast growth factor on cardiac fibroblasts grown under altered gravity conditions

    DEFF Research Database (Denmark)

    Ulbrich, Claudia; Leder, Annekatrin; Pietsch, Jessica

    2010-01-01

    Myocardium is very sensitive to gravitational changes. During a spaceflight cardiovascular atrophy paired with rhythm problems and orthostatic intolerance can occur. The aim of this study was to investigate the impact of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor...

  13. 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

  14. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  15. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    International Nuclear Information System (INIS)

    Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

    2014-01-01

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility

  16. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    -associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts...... could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular...

  17. Estradiol stimulation of inositolphospholipid metabolism in human endometrial fibroblasts

    International Nuclear Information System (INIS)

    Iida, K.; Imai, A.; Tamaya, T.

    1989-01-01

    Stimulated inositolphospholipid turnover has been proposed to constitute a signal-transducing mechanism in many cell types. To determine the inositolphospholipid turnover during stimulation by 17 beta-estradiol, the turnover kinetics of phospholipids was investigated in human endometrial fibroblasts. In cells incubated with [ 32 P] phosphate for 1 h, estradiol rapidly and persisitently (for at least 30 min) enhanced the rate of 32 P-labeling of phosphatidic acid (PA). On the other hand, after a lag time of 5 min, 32 P-labeling of phosphatidylinositol (PI) was also increased also. These sequential 32 P-labeling of PA and PI demonstrated that inositolphospholipid turnover was stimulated in fibroblasts exposed to estradiol. The rapid estrogen-stimulated inositolphospholipid turnover may not be through the mechanism associated with classical action of estrogen

  18. Bio-artificial pleura using an autologous dermal fibroblast sheet

    Science.gov (United States)

    Kanzaki, Masato; Takagi, Ryo; Washio, Kaoru; Kokubo, Mami; Yamato, Masayuki

    2017-10-01

    Air leaks (ALs) are observed after pulmonary resections, and without proper treatment, can produce severe complications. AL prevention is a critical objective for managing patients after pulmonary resection. This study applied autologous dermal fibroblast sheets (DFS) to close ALs. For sealing ALs in a 44-year-old male human patient with multiple bullae, a 5 × 15-mm section of skin was surgically excised. From this skin specimen, primary dermal fibroblasts were isolated and cultured for 4 weeks to produce DFSs that were harvested after a 10-day culture. ALs were completely sealed using surgical placement of these autologous DFSs. DFS were found to be a durable long-term AL sealant, exhibiting requisite flexibility, elasticity, durability, biocompatibility, and usability, resulting reliable AL closure. DFS should prove to be an extremely useful tissue-engineered pleura substitute.

  19. In vitro cytotoxicity of chemical preservatives on human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Daniel Gonsales Spindola

    2018-05-01

    Full Text Available ABSTRACT Preservatives are widely used substances that are commonly added to various cosmetic and pharmaceutical products to prevent or inhibit microbial growth. In this study, we compared the in vitro cytotoxicity of different types of currently used preservatives, including methylparaben, imidazolidinyl urea (IMU, and sodium benzoate, using the human newborn fibroblast cell line CCD1072Sk. Of the tested preservatives, only IMU induced a reduction in cell viability, as shown using the MTT assay and propidium iodide staining (IMU>methylparaben>sodium benzoate. IMU was shown to promote homeostatic alterations potentially related to the initiation of programed cell death, such as decreased mitochondrial membrane potential and caspase-3 activation, in the treated cells. Methylparaben and sodium benzoate were shown to have a very low cytotoxic activity. Taken together, our results suggest that IMU induces programed cell death in human fibroblasts by a canonical intrinsic pathway via mitochondrial perturbation and subsequent release of proapoptotic factors.

  20. Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

    International Nuclear Information System (INIS)

    Tatano, Yutaka; Fujinawa, Reiko; Kozutsumi, Yasunori; Takahashi, Tsutomu; Tsuji, Daisuke; Takeuchi, Naohiro; Tsuta, Kohji; Takada, Goro; Sakuraba, Hitoshi; Itoh, Kohji

    2008-01-01

    Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1β (IL-1β), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1β expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression

  1. Reduced superoxide dismutase activity in xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Nishigori, C.; Miyachi, Y.; Imamura, S.; Takebe, H.

    1989-01-01

    This study was performed in order to assess the possible protective effect of superoxide dismutase (SOD) on ultraviolet (UV) damage in xeroderma pigmentosum (XP) fibroblasts. SOD activity in fibroblasts originating from seven xeroderma pigmentosum (XP) patients was significantly lower than that in normal cells (p less than 0.005). Average SOD activity in XP cells belonging to complementation group A was 3.68 +/- 0.54 (n = 7) and that in normal human cells was 5.79 +/- 1.59 (n = 6). Addition of SOD before and during UV irradiation (UVB and UVC) to the cells caused no change in the amount of unscheduled DNA synthesis and UV survival. A possible involvement of reduced SOD in XP and a possible protective effect by SOD on UV damage is discussed

  2. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis; Goldmann, Wolfgang H.

    2009-01-01

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  3. EB1 is required for primary cilia assembly in fibroblasts

    DEFF Research Database (Denmark)

    Schrøder, Jacob M; Schneider, Linda; Christensen, Søren T

    2007-01-01

    EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and diff...... that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts....

  4. Fibroblasts and the extracellular matrix in right ventricular disease.

    Science.gov (United States)

    Frangogiannis, Nikolaos G

    2017-10-01

    Right ventricular failure predicts adverse outcome in patients with pulmonary hypertension (PH), and in subjects with left ventricular heart failure and is associated with interstitial fibrosis. This review manuscript discusses the cellular effectors and molecular mechanisms implicated in right ventricular fibrosis. The right ventricular interstitium contains vascular cells, fibroblasts, and immune cells, enmeshed in a collagen-based matrix. Right ventricular pressure overload in PH is associated with the expansion of the fibroblast population, myofibroblast activation, and secretion of extracellular matrix proteins. Mechanosensitive transduction of adrenergic signalling and stimulation of the renin-angiotensin-aldosterone cascade trigger the activation of right ventricular fibroblasts. Inflammatory cytokines and chemokines may contribute to expansion and activation of macrophages that may serve as a source of fibrogenic growth factors, such as transforming growth factor (TGF)-β. Endothelin-1, TGF-βs, and matricellular proteins co-operate to activate cardiac myofibroblasts, and promote synthesis of matrix proteins. In comparison with the left ventricle, the RV tolerates well volume overload and ischemia; whether the right ventricular interstitial cells and matrix are implicated in these favourable responses remains unknown. Expansion of fibroblasts and extracellular matrix protein deposition are prominent features of arrhythmogenic right ventricular cardiomyopathies and may be implicated in the pathogenesis of arrhythmic events. Prevailing conceptual paradigms on right ventricular remodelling are based on extrapolation of findings in models of left ventricular injury. Considering the unique embryologic, morphological, and physiologic properties of the RV and the clinical significance of right ventricular failure, there is a need further to dissect RV-specific mechanisms of fibrosis and interstitial remodelling. Published on behalf of the European Society of

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Tryptophan Transport in Human Fibroblast Cells—A Functional Characterization

    Directory of Open Access Journals (Sweden)

    Ravi Vumma

    2011-01-01

    Full Text Available There are indications that serotonergic neurotransmission is disturbed in several psychiatric disorders. One explanation may be disturbed transport of tryptophan (precursor for serotonin synthesis across cell membranes. Human fibroblast cells offer an advantageous model to study the transport of amino acids across cell membranes, since they are easy to propagate and the environmental factors can be controlled. The aim of this study was to functionally characterize tryptophan transport and to identify the main transporters of tryptophan in fibroblast cell lines from healthy controls. Tryptophan kinetic parameters ( V max and K m at low and high concentrations were measured in fibroblasts using the cluster tray method. Uptake of 3 H (5-L-tryptophan at different concentrations in the presence and absence of excess concentrations of inhibitors or combinations of inhibitors of amino acid transporters were also measured. Tryptophan transport at high concentration (0.5 mM had low affinity and high V max and the LAT1 isoform of system-L was responsible for approximately 40% of the total uptake of tryptophan. In comparison, tryptophan transport at low concentration (50 nM had higher affinity, lower V max and approximately 80% of tryptophan uptake was transported by system-L with LAT1 as the major isoform. The uptake of tryptophan at the low concentration was mainly sodium (Na + dependent, while uptake at high substrate concentration was mainly Na + independent. A series of different transporter inhibitors had varying inhibitory effects on tryptophan uptake. This study indicates that tryptophan is transported by multiple transporters that are active at different substrate concentrations in human fibroblast cells. The tryptophan transport trough system-L was mainly facilitated by the LAT1 isoform, at both low and high substrate concentrations of tryptophan.

  7. Analysis of primary cilia in directional cell migration in fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht

    2013-01-01

    summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration....... In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We...

  8. Radioprotective effect of catecholamines on the cultured Chinese hamster fibroblasts

    International Nuclear Information System (INIS)

    Chirkov, Yu.Yu.; Malatsidze, M.A.; Sobolev, A.S.

    1985-01-01

    On cultivated in vitro Chinese hamster fibroblasts radioprotective properties of adrenaline, noradrenaline and isoproterenol in different concentrations are studied. Isoproterenol radiopreventive effect is clearly manifested with its concentration being 1x10 -8 M; adrenaline and noradrenaline are efficient in higher concentrations. Propranolol, blocking β-adrenergic receptors, completely presents radioprotective effect of catecholamines on the cells. β-adrenergic mechanism of catecholamine radioprotective effect on Mammalia cells is discussed

  9. Fibroblastic osteosarcoma with epithelioid and squamous differentiation in a dog.

    Science.gov (United States)

    Jenkins, Tiffany L; Agnew, Dalen; Rissi, Daniel R

    2018-04-01

    A fibroblastic osteosarcoma with epithelioid and squamous differentiation in the distal femur of a 9-y-old spayed female Greyhound dog is described. Grossly, the tumor consisted of a pale-white, firm-to-hard mass that replaced the medullary and cortical areas of the distal end of the right femur. Histologically, the mass was composed predominantly of spindle cells admixed with areas of mineralized and non-mineralized osteoid matrix that were surrounded by stellate osteoblasts and scattered multinucleate giant cells, consistent with the diagnosis of a fibroblastic osteosarcoma. In addition, well-demarcated clusters of neoplastic epithelioid cells and foci of squamous differentiation with keratin pearls were present throughout the neoplasm. The spindle cells, epithelioid cells, and areas of squamous differentiation expressed cytoplasmic immunostaining for osteocalcin and osteonectin. The spindle cells and epithelioid cells were also immunopositive for vimentin. Epithelioid cells also expressed occasional cytoplasmic immunostaining for pancytokeratin (PCK) Lu-5, and areas of squamous differentiation were immunoreactive for PCK Lu-5 and high molecular weight CK; these areas were inconsistently immunoreactive for CK 5-6 and immunonegative for low molecular weight CK. Foci of squamous differentiation were not located within blood or lymphatic vessels, given that no immunoreactivity for factor VIII-related antigen was observed around these areas. A thorough autopsy and an evaluation of the medical history excluded a primary carcinoma or other neoplasm elsewhere in the dog. The findings were consistent with a diagnosis of fibroblastic osteosarcoma with epithelioid and squamous differentiation.

  10. Degradation of type IV collagen by neoplastic human skin fibroblasts

    International Nuclear Information System (INIS)

    Sheela, S.; Barrett, J.C.

    1985-01-01

    An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion

  11. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    International Nuclear Information System (INIS)

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E.

    1989-01-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings

  12. Potensi Terapeutik Fibroblast Growth Factor 21 terhadap Resistensi Insulin

    Directory of Open Access Journals (Sweden)

    Kurniasari Kurniasari

    2015-12-01

    Full Text Available Fibroblast growth factor 21 (FGF21 merupakan salah satu dari anggota FGF yang berperansebagai faktor endokrin. Hepar dan jaringan adiposa merupakan tempat kerja utama FGF21.Ekspresi FGF21 di hepar diatur oleh peroxisome proliferator activated receptor alpha (PPARαsedangkan di jaringan adiposa diatur oleh peroxisome proliferator activated receptor gamma(PPARγ. Kedua faktor transkripsi tersebut terlibat dalam metabolisme karbohidrat dan lipid. Padaresistensi insulin terdapat hiperglikemia, hiperinsulinemia, dan dislipidemia. Pemberian FGF21pada berbagai studi in vivo dan in vitro telah menunjukan potensi FGF21 dalam mengatasi kelainanakibat resistensi insulin sekaligus meningkatkan sensitivitas jaringan terhadap insulin. Kata kunci: FGF21, PPARγ, PPARα, resistensi insulin Fibroblast Growth Factor 21 (FGF21 Potension in InsulinResistance Treatment Abstract Fibroblast growth factor 21 (FGF21 is a member of FGF family that plays a role as endocrinefactor. Liver and adipose tissue are major target of FGF21. The expression of FGF21 in liveris regulated by peroxisome proliferator activated receptor alpha (PPARα, while peroxisomeproliferator activated receptor gamma (PPARγ regulate FGF21 expression in adipose tissue.Both transcription factors are involved in carbohydrate and lipid metabolism. Hyperglycemia,hyperinsulinemia, and dyslipidemia are observed in insulin resistance. Treatment with FGF21 inin vitro and in vivo study showed that FGF21 have the potential to overcome insulin resistance aswell as increasing tissue’s sensitivity towards insulin. Keywords: FGF21, PPARγ, PPARα, insulin resistance Normal 0 false false false IN X-NONE X-NONE

  13. Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

    International Nuclear Information System (INIS)

    Togo, Shinsaku; Polanska, Urszula M.; Horimoto, Yoshiya; Orimo, Akira

    2013-01-01

    Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies

  14. Effect of phenytoin and age on gingival fibroblast enzymes.

    Directory of Open Access Journals (Sweden)

    Surena Vahabi

    2014-06-01

    Full Text Available The alteration of cytokine balance is stated to exert greater influence on gingival overgrowth compared to the direct effect of the drug on the regulation of extracellular matrix metabolism. The current study evaluated the effect of phenytoin on the regulation of collagen, lysyl oxidase and elastin in gingival fibroblasts.Normal human gingival fibroblasts (HGFs were obtained from 4 healthy children and 4 adults. Samples were cultured with phenytoin. MTT test was used to evaluate the proliferation and ELISA was performed to determine the level of IL1β and PGE2 production by HGFs. Total RNA of gingival fibroblasts was extracted and RT-PCR was performed on samples. Mann-Whitney U test was used to analyze the data with an alpha error level less than 0.05.There was a significant difference in the expression of elastin between the controls and treated samples in both adult and pediatric groups and also in the lysyl oxidase expression of adult controls and treated adults. No significant difference was found between collagen expression in adults.The significant difference in elastin and lysyl oxidase expression between adult and pediatric samples indicates the significant effect of age on their production.

  15. Responses of fibroblasts and glial cells to nanostructured platinum surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

    2009-09-23

    The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

  16. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

    Directory of Open Access Journals (Sweden)

    Sara E. Howden

    2015-12-01

    Full Text Available The derivation of genetically modified induced pluripotent stem (iPS cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

  17. 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

  18. Thermoreversible gelation polymer as an embolic material for aneurysm treatment: a delivery device for dermal fibroblasts and basic fibroblast growing factor into experimental aneurysms in rats.

    Science.gov (United States)

    Dobashi, Hisashi; Akasaki, Yasuharu; Yuki, Ichiro; Arai, Takao; Ohashi, Hiroki; Murayama, Yuichi; Takao, Hiroyuki; Abe, Toshiaki

    2013-11-01

    This study evaluates whether thermoreversible gelation polymer (TGP) can be used as a delivery device to deploy dermal fibroblasts and cytokines into experimental aneurysms in rats. The right common iliac artery of rats was surgically ligated and an experimental aneurysm was created by applying exogenous elastase. Seven days later, two aneurysms were harvested and used as controls (Group A), two were embolized with pure TGP (Group B), two were embolized with TGP and basic fibroblast growth factor (bFGF) (Group C) and two were embolized with TGP loaded with rat dermal fibroblasts (Group D). The aneurysms were also embolized with TGP mixed with dermal fibroblasts and bFGF at different concentrations (10 ng/ml: Group E (n=2), 100 ng/ml: Group F (n=2), 1000 ng/ml: Group G (n=2)). Each aneurysm sample was harvested after 7 days and histologic analyses were performed. The most advanced thrombus organization in the aneurysm, such as prominent fibroblast proliferation and collagen deposition, was observed in Groups E, F and G, although there was no noticeable difference between the groups. Moderate thrombus organization was seen in Group D and minimal thrombus organization was seen in Groups B and C. TGP mixed with both dermal fibroblasts and bFGF induced the most advanced thrombus organization in the experimental aneurysms followed by TGP mixed only with dermal fibroblasts. TGP may be useful as a delivery device to deploy fibroblasts and cytokines into aneurysms.

  19. Studies of the in vivo radiosensitivity of human skin fibroblasts

    International Nuclear Information System (INIS)

    Hill, Richard P.; Kaspler, Pavel; Griffin, Anthony M.; O'Sullivan, Brian; Catton, Charles; Alasti, Hamideh; Abbas, Ahmar; Heydarian, Moustafa; Ferguson, Peter; Wunder, Jay S.; Bell, Robert S.

    2007-01-01

    Background and purpose: To examine the radiosensitivity of skin cells obtained directly from the irradiated skin of patients undergoing fractionated radiation treatment prior to surgery for treatment of soft tissue sarcoma (STS) and to determine if there was a relationship with the development of wound healing complications associated with the surgery post-radiotherapy. Methods: Micronucleus (MN) formation was measured in cells (primarily dermal fibroblasts) obtained from human skin at their first division after being removed from STS patients during post-radiotherapy surgery (2-9 weeks after the end of the radiotherapy). At the time of radiotherapy (planned tumor dose - 50 Gy in 25 daily fractions) measurements were made of surface skin dose at predetermined marked sites. Skin from these sites was obtained at surgery and cell suspensions were prepared directly for the cytokinesis-blocked MN assay. Cultured strains of the fibroblasts were also established from skin nominally outside the edge of the radiation beam and DNA damage (MN formation) was examined following irradiation in vitro for comparison with the results from the in situ irradiations. Results: Extensive DNA damage (MN) was detectable in fibroblasts from human skin at extended periods after irradiation (2-9 weeks after the end of the 5-week fractionated radiotherapy). Analysis of skin receiving a range of doses demonstrated that the level of damage observed was dose dependent. There was no clear correlation between the level of damage observed after irradiation in situ and irradiation of cell strains in culture. Similarly, there was no correlation between the extent of MN formation following in situ irradiation and the propensity for the patient to develop wound healing complications post-surgery. Conclusions: Despite the presence of DNA damage in dermal fibroblasts weeks after the end of the radiation treatment, there was no relationship between this damage and wound healing complications following

  20. Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM.

    Science.gov (United States)

    Lemieux, Maxime; Laflamme, Olivier D; Thiry, Louise; Boulanger-Piette, Antoine; Frenette, Jérôme; Bretzner, Frédéric

    2016-03-01

    Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM(2J) mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM(2J) mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM(2J) mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits. Copyright © 2016 the American Physiological Society.

  1. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    International Nuclear Information System (INIS)

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J.

    1990-01-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation

  2. Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

    Science.gov (United States)

    Philippeos, Christina; Telerman, Stephanie B; Oulès, Bénédicte; Pisco, Angela O; Shaw, Tanya J; Elgueta, Raul; Lombardi, Giovanna; Driskell, Ryan R; Soldin, Mark; Lynch, Magnus D; Watt, Fiona M

    2018-04-01

    Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

    International Nuclear Information System (INIS)

    Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

    2005-01-01

    The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

  4. Effect of storage media on the proliferation of periodontal ligament fibroblasts

    International Nuclear Information System (INIS)

    Lauer, H.C.; Mueller, J.G.; Gross, J.; Horster, M.F.

    1987-01-01

    The effect of storage media, which are routinely used in replantation, upon the proliferative capacity of periodontal ligament fibroblasts, was compared with the effect of a tissue culture medium. The periodontal tissue was obtained from mandibular central incisors of White New Zealand rabbits. The experiments were performed in fibroblasts derived during second subculture. The storage media were physiologic salt solution, Ringer's solution and Rivanol; the tissue culture medium was alpha-minimum essential medium without nucleosides. The incubation period was 1 hour. [ 3 H]-thymidine incorporation and cell counts were taken to indicate changes in the proliferative capacity of the fibroblasts. The tissue culture experiments showed that the proliferative ability of the periodontal ligament fibroblasts was dependent upon the composition of the storage medium. Physiologic salt solution, Ringer's solution and Rivanol were unable to maintain the metabolism of the fibroblasts. alpha-MEM medium, however, was capable of stimulating proliferation of the periodontal ligament fibroblasts

  5. Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

    Science.gov (United States)

    Tesco, G; Vergelli, M; Grassilli, E; Salomoni, P; Bellesia, E; Sikora, E; Radziszewska, E; Barbieri, D; Latorraca, S; Fagiolo, U; Santacaterina, S; Amaducci, L; Tiozzo, R; Franceschi, C; Sorbi, S

    1998-03-27

    Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

  6. Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Jaroslaw Suchanski

    Full Text Available In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first

  7. Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

    Science.gov (United States)

    Suchanski, Jaroslaw; Tejchman, Anna; Zacharski, Maciej; Piotrowska, Aleksandra; Grzegrzolka, Jedrzej; Chodaczek, Grzegorz; Nowinska, Katarzyna; Rys, Janusz; Dziegiel, Piotr; Kieda, Claudine; Ugorski, Maciej

    2017-01-01

    In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such

  8. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

    International Nuclear Information System (INIS)

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

    2008-01-01

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2 -/- ) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2 -/- MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2 -/- MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2 -/- MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts

  9. Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair.

    Science.gov (United States)

    Chiquet, Matthias; Katsaros, Christos; Kletsas, Dimitris

    2015-06-01

    Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal-like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound-healing processes of the individual patient. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes

    OpenAIRE

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation...

  11. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2014-08-01

    Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  12. PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

    Science.gov (United States)

    Khamaisi, Mogher; Katagiri, Sayaka; Keenan, Hillary; Park, Kyoungmin; Maeda, Yasutaka; Li, Qian; Qi, Weier; Thomou, Thomas; Eschuk, Danielle; Tellechea, Ana; Veves, Aris; Huang, Chenyu; Orgill, Dennis Paul; Wagers, Amy; King, George L

    2016-03-01

    Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

  13. Fibroblast implantation enhances wound healing as indicated by breaking strength determinations

    Energy Technology Data Exchange (ETDEWEB)

    Krueger, W W; Goepfert, H; Romsdahl, M; Hersen, J; Withers, R H; Jesse, R H

    1978-09-01

    Irradiation of normal tissues at the dose/time factor employed in the treatment of solid tumors impairs the subsequent healing of surgical wounds made in those tissues. Irreversible radiation damage to regional fibroblasts is one cause of impared healing. This study was conducted to determine whether syngeneic guinea pig fibroblasts is one cause of impared healing. This study was conducted to determine whether syngeneic guinea pig fibroblasts, harvested from tissue culture when injected into irradiated guinea pig skin at the time of wound closure, could improve wound healing. Breaking strength determinations indicate that irradiated wounds demonstrate enhanced wound healing if implanted with fibroblasts.

  14. Biological Differences between Hanwoo longissimus dorsi and semimembranosus Muscles in Collagen Synthesis of Fibroblasts.

    Science.gov (United States)

    Subramaniyan, Sivakumar Allur; Hwang, Inho

    2017-01-01

    Variations in physical toughness between muscles and animals are a function of growth rate and extend of collagen type I and III. The current study was designed to investigate the ability of growth rate, collagen concentration, collagen synthesizing and degrading genes on two different fibroblast cells derived from Hanwoo m. longissimus dorsi (LD) and semimembranosus (SM) muscles. Fibroblast cell survival time was determined for understanding about the characteristics of proliferation rate between the two fibroblasts. We examined the collagen concentration and protein expression of collagen type I and III between the two fibroblasts. The mRNA expression of collagen synthesis and collagen degrading genes to elucidate the molecular mechanisms on toughness and tenderness through collagen production between the two fibroblast cells. From our results the growth rate, collagen content and protein expression of collagen type I and III were significantly higher in SM than LD muscle fibroblast. The mRNA expressions of collagen synthesized genes were increased whereas the collagen degrading genes were decreased in SM than LD muscle. Results from confocal microscopical investigation showed increased fluorescence of collagen type I and III appearing stronger in SM than LD muscle fibroblast. These results implied that the locomotion muscle had higher fibroblast growth rate, leads to produce more collagen, and cause tougher than positional muscle. This in vitro study mirrored that background toughness of various muscles in live animal is likely associated with fibroblast growth pattern, collagen synthesis and its gene expression.

  15. Pearl extract enhances the migratory ability of fibroblasts in a wound healing model.

    Science.gov (United States)

    Li, Yi-Chen; Chen, Chi-Ruei; Young, Tai-Horng

    2013-03-01

    For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 μg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. Medium containing PL (300 μg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.

  16. Magnitude and duration of stretch modulate fibroblast remodeling.

    Science.gov (United States)

    Balestrini, Jenna L; Billiar, Kristen L

    2009-05-01

    Mechanical cues modulate fibroblast tractional forces and remodeling of extracellular matrix in healthy tissue, healing wounds, and engineered matrices. The goal of the present study is to establish dose-response relationships between stretch parameters (magnitude and duration per day) and matrix remodeling metrics (compaction, strength, extensibility, collagen content, contraction, and cellularity). Cyclic equibiaxial stretch of 2-16% was applied to fibroblast-populated fibrin gels for either 6 h or 24 h/day for 8 days. Trends in matrix remodeling metrics as a function of stretch magnitude and duration were analyzed using regression analysis. The compaction and ultimate tensile strength of the tissues increased in a dose-dependent manner with increasing stretch magnitude, yet remained unaffected by the duration in which they were cycled (6 h/day versus 24 h/day). Collagen density increased exponentially as a function of both the magnitude and duration of stretch, with samples stretched for the reduced duration per day having the highest levels of collagen accumulation. Cell number and failure tension were also dependent on both the magnitude and duration of stretch, although stretch-induced increases in these metrics were only present in the samples loaded for 6 h/day. Our results indicate that both the magnitude and the duration per day of stretch are critical parameters in modulating fibroblast remodeling of the extracellular matrix, and that these two factors regulate different aspects of this remodeling. These findings move us one step closer to fully characterizing culture conditions for tissue equivalents, developing improved wound healing treatments and understanding tissue responses to changes in mechanical environments during growth, repair, and disease states.

  17. Fibroblast adhesion and activation onto micro-machined titanium surfaces.

    Science.gov (United States)

    Guillem-Marti, J; Delgado, L; Godoy-Gallardo, M; Pegueroles, M; Herrero, M; Gil, F J

    2013-07-01

    Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 μm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 μm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (fibrotic response. © 2012 John Wiley & Sons A/S.

  18. Cellular radiosensitivity and DNA damage in primary human fibroblasts

    International Nuclear Information System (INIS)

    Wurm, R.; Burnet, N.G.; Duggal, N.

    1994-01-01

    To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

  19. Platelets stimulate fibroblast-mediated contraction of collagen gels

    Directory of Open Access Journals (Sweden)

    Lundahl Joachim

    2003-10-01

    Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

  20. DNA synthesis in vitro in human fibroblast preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, W.K.

    1983-01-01

    When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

  1. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    Science.gov (United States)

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  2. 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.

  3. Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

    International Nuclear Information System (INIS)

    Chan, G.L.; Little, J.B.

    1979-01-01

    Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhancement of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells. (orig.)

  4. Nonmuscle myosin IIA and IIB differentially contribute to intrinsic and directed migration of human embryonic lung fibroblasts.

    Science.gov (United States)

    Kuragano, Masahiro; Murakami, Yota; Takahashi, Masayuki

    2018-03-25

    Nonmuscle myosin II (NMII) plays an essential role in directional cell migration. In this study, we investigated the roles of NMII isoforms (NMIIA and NMIIB) in the migration of human embryonic lung fibroblasts, which exhibit directionally persistent migration in an intrinsic manner. NMIIA-knockdown (KD) cells migrated unsteadily, but their direction of migration was approximately maintained. By contrast, NMIIB-KD cells occasionally reversed their direction of migration. Lamellipodium-like protrusions formed in the posterior region of NMIIB-KD cells prior to reversal of the migration direction. Moreover, NMIIB KD led to elongation of the posterior region in migrating cells, probably due to the lack of load-bearing stress fibers in this area. These results suggest that NMIIA plays a role in steering migration by maintaining stable protrusions in the anterior region, whereas NMIIB plays a role in maintenance of front-rear polarity by preventing aberrant protrusion formation in the posterior region. These distinct functions of NMIIA and NMIIB might promote intrinsic and directed migration of normal human fibroblasts. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

    Science.gov (United States)

    Jaquemar, D; Schenker, T; Trueb, B

    1999-03-12

    We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

  6. 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

  7. 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

  8. Effect of chloramphenicol on sister chromatid exchange in bovine fibroblasts.

    Science.gov (United States)

    Arruga, M V; Catalan, J; Moreno, C

    1992-03-01

    The genotoxic potential of different chloramphenicol concentrations (5, 20, 40 and 60 micrograms ml-1) was investigated in bovine fibroblast primary lines by sister chromatid exchange assay. Chloramphenicol acted for long enough to ensure similar effects to persistent storage in the kidney. In this experiment 10 micrograms ml-1 of 5-bromodeoxyuridine was added for 60 hours for all doses of chloramphenicol and to the control. When the tissue culture cells were exposed to increasing doses, increased numbers of sister chromatid exchanges developed. Differences were significantly different to the control.

  9. Cancer Associated Fibroblasts and Tumor Growth: Focus on Multiple Myeloma

    International Nuclear Information System (INIS)

    De Veirman, Kim; Rao, Luigia; De Bruyne, Elke; Menu, Eline; Van Valckenborgh, Els; Van Riet, Ivan; Frassanito, Maria Antonia; Di Marzo, Lucia; Vacca, Angelo; Vanderkerken, Karin

    2014-01-01

    Cancer associated fibroblasts (CAFs) comprise a heterogeneous population that resides within the tumor microenvironment. They actively participate in tumor growth and metastasis by production of cytokines and chemokines, and the release of pro-inflammatory and pro-angiogenic factors, creating a more supportive microenvironment. The aim of the current review is to summarize the origin and characteristics of CAFs, and to describe the role of CAFs in tumor progression and metastasis. Furthermore, we focus on the presence of CAFs in hypoxic conditions in relation to multiple myeloma disease

  10. Cancer Associated Fibroblasts and Tumor Growth: Focus on Multiple Myeloma

    Energy Technology Data Exchange (ETDEWEB)

    De Veirman, Kim, E-mail: kdeveirm@vub.ac.be [Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels 1090 (Belgium); Rao, Luigia [Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels 1090 (Belgium); Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine, University of Bari Medical School, Bari I-70124 (Italy); De Bruyne, Elke; Menu, Eline; Van Valckenborgh, Els [Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels 1090 (Belgium); Van Riet, Ivan [Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels 1090 (Belgium); Stem Cell Laboratory, Division of Clinical Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels 1090 (Belgium); Frassanito, Maria Antonia [Department of Biomedical Sciences and Human Oncology, Section of General Pathology, University of Bari Medical School, Bari I-70124 (Italy); Di Marzo, Lucia; Vacca, Angelo [Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine, University of Bari Medical School, Bari I-70124 (Italy); Vanderkerken, Karin, E-mail: kdeveirm@vub.ac.be [Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels 1090 (Belgium)

    2014-06-27

    Cancer associated fibroblasts (CAFs) comprise a heterogeneous population that resides within the tumor microenvironment. They actively participate in tumor growth and metastasis by production of cytokines and chemokines, and the release of pro-inflammatory and pro-angiogenic factors, creating a more supportive microenvironment. The aim of the current review is to summarize the origin and characteristics of CAFs, and to describe the role of CAFs in tumor progression and metastasis. Furthermore, we focus on the presence of CAFs in hypoxic conditions in relation to multiple myeloma disease.

  11. 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

  12. Effect of microemulsions on cell viability of human dermal fibroblasts

    Science.gov (United States)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  13. Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma.

    Science.gov (United States)

    Le, Tran; New, Jacob; Jones, Joel W; Usman, Shireen; Yalamanchali, Sreeya; Tawfik, Ossama; Hoover, Larry; Bruegger, Dan E; Thomas, Sufi Mary

    2017-10-01

    Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA. © 2017 ARS

  14. MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

    Science.gov (United States)

    Wang, Daren; Zhang, Hui; Li, Min; Frid, Maria G; Flockton, Amanda R; McKeon, B Alexandre; Yeager, Michael E; Fini, Mehdi A; Morrell, Nicholas W; Pullamsetti, Soni S; Velegala, Sivareddy; Seeger, Werner; McKinsey, Timothy A; Sucharov, Carmen C; Stenmark, Kurt R

    2014-01-03

    Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly

  15. 10 CFR 503.21 - Lack of alternate fuel supply.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for New Facilities § 503.21 Lack of alternate fuel supply. (a) Eligibility. Section 211(a)(1) of the Act provides for...

  16. Response of human corneal fibroblasts on silk film surface patterns.

    Science.gov (United States)

    Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L

    2010-06-11

    Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.

  17. Effect of saccharin on metabolic cooperation between human fibroblasts

    International Nuclear Information System (INIS)

    Mosser, D.D.; Bols, N.C.

    1983-01-01

    Autoradiography was used to study the effect of saccharin on metabolic cooperation between human diploid fibroblasts. When the donors, HGPRT+ cells, and recipients, HGPRT- cells, were plated together in the presence of saccharin, all the interactions that developed in 4 and 24 h were positive for metabolic cooperation. When saccharin was added after donor cells and recipient cells had made contact, the proportion of interactions that were positive for metabolic cooperation was unchanged but the number of grains over primary recipients was reduced. However, in donor cells saccharin caused a reduction in [ 3 H]hypoxanthine incorporation into both acid-soluble and acid-insoluble fractions, although the relative distribution of radioactivity between these two fractions and between the phosphorylated and non-phosphorylated derivatives of [ 3 H]hypoxanthine was unchanged. Metabolic cooperation was studied under conditions in which the number of grains over the nuclei of both the primary recipient and the primary recipient's donor could be counted. The change in the number of grains over these two cell types in response to saccharin was compared and found to be the same. Thus in normal human fibroblasts saccharin does not appear to affect metabolic cooperation, which is a measure of cell-to-cell communication

  18. CD14-negative isolation enhances chondrogenesis in synovial fibroblasts.

    Science.gov (United States)

    Bilgen, Bahar; Ren, Yuexin; Pei, Ming; Aaron, Roy K; Ciombor, Deborah McK

    2009-11-01

    Synovial membrane has been shown to contain mesenchymal stem cells. We hypothesized that an enriched population of synovial fibroblasts would undergo chondrogenic differentiation and secrete cartilage extracellular matrix to a greater extent than would a mixed synovial cell population (MSCP). The optimum doses of transforming growth factor beta 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1) for chondrogenesis were investigated. CD14-negative isolation was used to obtain a porcine cell population enriched in type-B synovial fibroblasts (SFB) from an MSCP. The positive cell surface markers in SFB were CD90, CD44, and cadherin-11. SFB and MSCP were cultured in the presence of 20 ng/mL TGF-beta1 for 7 days, and SFB were demonstrated to have higher chondrogenic potential. Further dose-response studies were carried out using the SFB cells and several doses of TGF-beta1 (2, 10, 20, and 40 ng/mL) and/or IGF-1 (1, 10, 100, and 500 ng/mL) for 14 days. TGF-beta1 supplementation was essential for chondrogenesis and prevention of cell death, whereas IGF-1 did not have a significant effect on the SFB cell number or glycosaminoglycan production. This study demonstrates that the CD14-negative isolation yields an enhanced cell population SFB that is more potent than MSCP as a cell source for cartilage tissue engineering.

  19. Differential Gene Expression of Fibroblasts: Keloid versus Normal

    Directory of Open Access Journals (Sweden)

    Michael F. Angel

    2002-11-01

    Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

  20. Protein Adsorption and Subsequent Fibroblasts Adhesion on Hydroxyapatite Nanocrystals

    International Nuclear Information System (INIS)

    Tagaya, Motohiro; Ikoma, Toshiyuki; Yoshioka, Tomohiko; Tanaka, Junzo; Takemura, Taro; Hanagata, Nobutaka

    2011-01-01

    Quartz crystal microbalance with dissipation (QCM-D) technique was employed for protein adsorption and subsequent fibroblast adhesion on hydroxyapatite (HAp) nanocrystals. The pre-adsorption of three proteins (albumin (BSA) or fibronectin (Fn) or collagen (Col)) and subsequent adsorption of fetal bovine serum (FBS), and the adhesion of fibroblasts on the surface were in situ monitored, and evaluated with the frequency shift (Δf) and dissipation energy shift (ΔD), and the viscoelastic change as ΔD-Δf plot. The Col adsorption showed larger Δf and ΔD values compared with BSA or Fn adsorption, and the subsequent FBS adsorption depended on the pre-adsorbed proteins. The ΔD-Δf plot of the cell adhesion also showed the different behaviour on the surfaces, indicating the process affected by cell-protein interactions. The confocal laser scanning microscope images of adherent cells showed the different morphology and pseudopod on the surfaces. The cells adhered on the surfaces modified with Fn and Col had the uniaxially expanded shape with fibrous pseudopods, while those modified with BSA had round shape. The different cell-protein interaction would cause the arrangement of extracellular matrix and cytoskeleton changes at the interfaces.

  1. Cytotoxic effects of nickel nanowires in human fibroblasts

    KAUST Repository

    Felix Servin, Laura P.

    2016-03-09

    The increasing interest in the use of magnetic nanostructures for biomedical applications necessitates rigorous studies to be carried out in order to determine their potential toxicity. This work attempts to elucidate the cytotoxic effects of nickel nanowires (NWs) in human fibroblasts WI-38 by a colorimetric assay (MTT) under two different parameters: NW concentration and exposure time. This was complemented with TEM and confocal images to assess the NWs internalization and to identify any changes in the cell morphology. Ni NWs were fabricated by electrodeposition using porous alumina templates. Energy dispersive X-Ray analysis, scanning electron microscopy and transmission electron microscopy imaging were used for NW characterization. The results showed decreased cell metabolic activity for incubation times longer than 24 hours and no negative effects for exposure times shorter than that. The cytotoxicity effects for human fibroblasts were then compared with those reported for HCT 116 cells, and the findings point out that it is relevant to consider the cellular size. In addition, the present study compares the toxic effects of equivalent amounts of nickel in the form of its salt to those of NWs and shows that the NWs are more toxic than the salts. Internalized NWs were found in vesicles inside of the cells where their presence induced inflammation of the endoplasmic reticulum.

  2. Age related changes in steroid receptors on cultured lung fibroblasts

    International Nuclear Information System (INIS)

    Barile, F.A.; Bienkowski, R.S.

    1986-01-01

    The number of high affinity glucocorticoid receptors (Ro) on human fetal lung fibroblasts decreases as the cells age in vitro, and it has been suggested that these cell systems may be useful models of age-related changes in vivo. They examined the relation between change in Ro with in vitro aging and donor age. Confluent monolayers of lung fibroblasts at various population doubling levels (PDL), were incubated with ( 3 H)-dexamethasone (( 3 H)Dex) either alone or with excess (.01 mM) Dex. Specific binding was calculated as the difference between radioactivity in cells incubated with and without unlabeled Dex; Scatchard plots were used to analyze the data. Ro, measured as fmol ( 3 H)Dex/10 6 cells, for two lines of human fetal cells (HFL-1 and MRC-5) decreased with increasing age in vitro. However, human newborn (CRL-1485) and adult (CCL-201) cells and fetal rabbit cells (FAB-290), showed increases in Ro with continuous passage. For each cell line, the affinity constant (K/sub d/) did not change significantly with passage. They conclude that the direction of changes in steroid receptor levels on cells aging in vitro is influenced by donor age and species. Caution should be used in applying results obtained from model systems to aging organisms

  3. Effect of Arctium lappa (burdock) extract on canine dermal fibroblasts.

    Science.gov (United States)

    Pomari, Elena; Stefanon, Bruno; Colitti, Monica

    2013-12-15

    Although the biological activities of Arctium lappa (burdock) have been already investigated in human and other species, data evaluating the molecular mechanisms have not been reported in the dog. In this study we analyzed for the first time the effect of a root extract of burdock on molecular responses in canine dermal fibroblasts with H2O2 stimulation (H group), with burdock treatment (B group) and with H2O2 stimulation and burdock treatment (BH group), using RNAseq technology. Differentially expressed genes (P<0.05) of H, B and BH groups in comparison to the untreated sample (negative control, C group) were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). The expression profile of canine dermal fibroblasts treated with burdock extract with or without H2O2 stimulation, showed an up-regulation of mitochondrial superoxide dismutase (SOD2), disheveled 3 (DVL3) and chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGALNACT2). The data suggested that burdock has implications in cell adhesion and gene expression with the modulation of Wnt/β catenin signaling and Chondroitin Sulphate Biosynthesis that are particularly important for the wound healing process. © 2013 Elsevier B.V. All rights reserved.

  4. Biochemical changes to fibroblast cells subjected to ionizing radiation.

    Science.gov (United States)

    Jones, Pamala; Benghuzzi, Hamed; Tucci, Michelle; Richards, Latoya; Harrison, George; Patel, Ramesh

    2008-01-01

    High energy X-rays are capable of interacting with biological membranes to cause both functional and structural modifications. The goal of the present study was to investigate the effects human fibroblast cells exposed multiple times to 10 Gy over time. Following exposures of 2, 3, or 4 times to 10 Gy/10min the cells were evaluated for cell number changes, membrane damage, and intracellular glutathione content after 24, 48 and 72 hours. Twenty-four hours following exposure the cell numbers were reduced and increased levels of cellular membrane damage was evident. This trend was observed for the duration of the study. Interestingly, there was not an exposure dependent increase in cell damage or cell loss with time. Intracellular antioxidant systems were activated as indicated by anincrease in total cellular glutathione content. Additional studies are needed to determine if the cellular reduction is caused by a direct effect of the X-rays targeting the DNA or an indirect effect of the X-ray targeting the cellular membrane, which then generates radicals that target cell cycle checkpoints or DNA damage. In conclusion, fibroblast cells can be used to determine early and late events of cellular function following exposure to harmful levels of radiation exposure and results of exposure can be seen within twenty four hours.

  5. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

    Directory of Open Access Journals (Sweden)

    Emond Mary

    2007-09-01

    Full Text Available Abstract Background Marfan syndrome (MFS is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value -6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status. An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

  6. Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts.

    Science.gov (United States)

    Jędrak, Paulina; Krygier, Magdalena; Tońska, Katarzyna; Drozd, Małgorzata; Kaliszewska, Magdalena; Bartnik, Ewa; Sołtan, Witold; Sitek, Emilia J; Stanisławska-Sachadyn, Anna; Limon, Janusz; Sławek, Jarosław; Węgrzyn, Grzegorz; Barańska, Sylwia

    2017-08-01

    Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

  7. Protein Adsorption and Subsequent Fibroblasts Adhesion on Hydroxyapatite Nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Tagaya, Motohiro; Ikoma, Toshiyuki; Yoshioka, Tomohiko; Tanaka, Junzo [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, Tokyo, Tokyo 152-8550 (Japan); Takemura, Taro; Hanagata, Nobutaka, E-mail: tagaya.m.aa@m.titech.ac.jp [Biomaterials Center, National Institute for Materials Science, Tsukuba, Ibaraki 305-0047 (Japan)

    2011-10-29

    Quartz crystal microbalance with dissipation (QCM-D) technique was employed for protein adsorption and subsequent fibroblast adhesion on hydroxyapatite (HAp) nanocrystals. The pre-adsorption of three proteins (albumin (BSA) or fibronectin (Fn) or collagen (Col)) and subsequent adsorption of fetal bovine serum (FBS), and the adhesion of fibroblasts on the surface were in situ monitored, and evaluated with the frequency shift ({Delta}f) and dissipation energy shift ({Delta}D), and the viscoelastic change as {Delta}D-{Delta}f plot. The Col adsorption showed larger {Delta}f and {Delta}D values compared with BSA or Fn adsorption, and the subsequent FBS adsorption depended on the pre-adsorbed proteins. The {Delta}D-{Delta}f plot of the cell adhesion also showed the different behaviour on the surfaces, indicating the process affected by cell-protein interactions. The confocal laser scanning microscope images of adherent cells showed the different morphology and pseudopod on the surfaces. The cells adhered on the surfaces modified with Fn and Col had the uniaxially expanded shape with fibrous pseudopods, while those modified with BSA had round shape. The different cell-protein interaction would cause the arrangement of extracellular matrix and cytoskeleton changes at the interfaces.

  8. Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

    Science.gov (United States)

    Galetzka, D; Weis, E; Rittner, G; Schindler, D; Haaf, T

    2008-01-01

    Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development. (c) 2008 S. Karger AG, Basel.

  9. 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.

  10. Heterogeneity in Fibroblast Proliferation and Survival in Idiopathic Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    David Michael Habiel

    2014-01-01

    Full Text Available Idiopathic pulmonary fibrosis (IPF is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. Myofibroblasts have been shown to arise from interstitial fibroblasts, epithelial to mesenchymal transition of type II alveolar epithelial cells, and the differentiation of recruited fibrocytes. There are many mechanisms that are utilized by these cells for survival, proliferation and persistent activation including up-regulation of cytokines (i.e. Interlukin 6 (IL-6, cytokine receptors (i.e. Interlukin 6 Receptor 1 (IL-6R1, Glycoprotein 130 (gp130 and C-C Chemokine Receptor type 7 (CCR7 and innate pattern recognition receptors (PRRs; i.e. Toll Like Receptor 9 (TLR9. In this review, we will discuss the role of the cytokines IL-6 and CCL21, their receptors and the pattern recognition receptor (PRR, TLR9, in fibroblast recruitment, activation, survival and differentiation into myofibroblasts in IPF.

  11. [Nuclear matrix organization of the chromocenters in cultured murine fibroblasts].

    Science.gov (United States)

    Sheval', E V; Poliakov, V Iu

    2010-01-01

    In the current work, the structural organization of nuclear matrix of pericentromeric heterochromatin blocks (chromocenters) inside cultured murine fibroblasts was investigated. After 2 M NaCl extraction without DNase I treatment, chromocenters were extremely swelled, and it was impossible to detect them using conventional electron microscopy. Using immunogolding with anti-topoisomerase IIalpha antibody, we demonstrated that residual chromocenters were subdivided into numerous discrete aggregates. After 2 M NaCl extraction with DNase I treatment, the residual chromocenters appeared as a dense meshwork of thin fibers, and using this feature, the residual chromocenters were easily distinguished from the rest of nuclear matrix. After extraction with dextran sulfate and heparin, the chromocenters were decondensed, and chromatin complexes having rosette organization (central core from which numerous DNA fibers radiated) were seen. Probably, the appearance of these rosettes was a consequence of incomplete chromatin extraction. Thus, the nuclear matrix of pericentromeric chromosome regions in cultured murine fibroblasts differs morphologically from the rest of nuclear matrix.

  12. Three-Dimensional Human Tissue Models That Incorporate Diabetic Foot Ulcer-Derived Fibroblasts Mimic In Vivo Features of Chronic Wounds

    Science.gov (United States)

    Maione, Anna G.; Brudno, Yevgeny; Stojadinovic, Olivera; Park, Lara K.; Smith, Avi; Tellechea, Ana; Leal, Ermelindo C.; Kearney, Cathal J.; Veves, Aristidis; Tomic-Canic, Marjana; Mooney, David J.

    2015-01-01

    Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell–cell and cell–matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications. PMID:25343343

  13. The effect of polarized light on the organization of collagen secreted by fibroblasts.

    Science.gov (United States)

    Akilbekova, Dana; Boddupalli, Anuraag; Bratlie, Kaitlin M

    2018-04-01

    Recent studies have demonstrated the beneficial effect of low-power lasers and polarized light on wound healing, inflammation, and the treatment of rheumatologic and neurologic disorders. The overall effect of laser irradiation treatment is still controversial due to the lack of studies on the biochemical mechanisms and the optimal parameters for the incident light that should be chosen for particular applications. Here, we study how NIH/3T3 fibroblasts respond to irradiation with linearly polarized light at different polarization angles. In particular, we examined vascular endothelial growth factor (VEGF) secretion, differentiation to myofibroblasts, and collagen organization in response to 800 nm polarized light at 0°, 45°, 90°, and 135° with a power density of 40 mW/cm 2 for 6 min every day for 6 days. Additional experiments were conducted in which the polarization angle of the incident was changed every day to induce an isotropic distribution of collagen. The data presented here shows that polarized light can upregulate VEGF production, myofibroblast differentiation, and induce different collagen organization in response to different polarization angles of the incident beam. These results are encouraging and demonstrate possible methods for controlling cell response through the polarization angle of the laser light, which has potential for the treatment of wounds.

  14. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    International Nuclear Information System (INIS)

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

    2005-01-01

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

  15. Central Fibroblast Growth Factor 21 Browns White Fat via Sympathetic Action in Male Mice.

    Science.gov (United States)

    Douris, Nicholas; Stevanovic, Darko M; Fisher, Ffolliott M; Cisu, Theodore I; Chee, Melissa J; Nguyen, Ngoc L; Zarebidaki, Eleen; Adams, Andrew C; Kharitonenkov, Alexei; Flier, Jeffrey S; Bartness, Timothy J; Maratos-Flier, Eleftheria

    2015-07-01

    Fibroblast growth factor 21 (FGF21) has multiple metabolic actions, including the induction of browning in white adipose tissue. Although FGF21 stimulated browning results from a direct interaction between FGF21 and the adipocyte, browning is typically associated with activation of the sympathetic nervous system through cold exposure. We tested the hypothesis that FGF21 can act via the brain, to increase sympathetic activity and induce browning, independent of cell-autonomous actions. We administered FGF21 into the central nervous system via lateral ventricle infusion into male mice and found that the central treatment increased norepinephrine turnover in target tissues that include the inguinal white adipose tissue and brown adipose tissue. Central FGF21 stimulated browning as assessed by histology, expression of uncoupling protein 1, and the induction of gene expression associated with browning. These effects were markedly attenuated when mice were treated with a β-blocker. Additionally, neither centrally nor peripherally administered FGF21 initiated browning in mice lacking β-adrenoceptors, demonstrating that an intact adrenergic system is necessary for FGF21 action. These data indicate that FGF21 can signal in the brain to activate the sympathetic nervous system and induce adipose tissue thermogenesis.

  16. DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure

    International Nuclear Information System (INIS)

    Focke, Frauke; Schuermann, David; Kuster, Niels; Schaer, Primo

    2010-01-01

    Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage.

  17. DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure

    Energy Technology Data Exchange (ETDEWEB)

    Focke, Frauke; Schuermann, David [Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel (Switzerland); Kuster, Niels [IT' IS Foundation, Zeughausstrasse 43, CH-8004 Zurich (Switzerland); Schaer, Primo, E-mail: primo.schaer@unibas.ch [Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel (Switzerland)

    2010-01-05

    Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage.

  18. Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

    Science.gov (United States)

    Pasi, Francesca; Sanna, Samuele; Paolini, Alessandro; Alquati, Marco; Lascialfari, Alessandro; Corti, Maurizio Enrico; Liberto, Riccardo Di; Cialdai, Francesca; Monici, Monica; Nano, Rosanna

    2016-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

  19. Comparison between the radiosensitivity of human, mouse and chicken fibroblast-like cells using short-term endpoints

    International Nuclear Information System (INIS)

    Diatloff-Zito, C.; Loria, E.; Maciera-Coelho, A.; Deschavanne, P.J.; Malaise, E.P.

    1981-01-01

    A comparative study has been made of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells. (author)

  20. FRS2α is Essential for the Fibroblast Growth Factor to Regulate the mTOR Pathway and Autophagy in Mouse Embryonic Fibroblasts

    OpenAIRE

    Xiang Lin, Yongyou Zhang, Leyuan Liu, Wallace L. McKeehan, Yuemao Shen, Siyang Song, Fen Wang

    2011-01-01

    Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/A...

  1. 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

  2. High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Xiaoying [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Xu, Xingbo [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Zeisberg, Elisabeth M. [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany); Zeisberg, Michael, E-mail: mzeisberg@med.uni-goettingen.de [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany)

    2016-04-08

    Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones. - Highlights: • We exposed human kidney fibroblasts to media containing 1 mM or 3 mM phosphate. • Increased phosphate influx causes phosphorylation of DNA methyltransferase Dnmt1. • Phosphorylated Dnmt1 causes promoter methylation and transcriptional silencing of RASAL1. • Depletion of RASAL1 causes increased intrinsic Ras-GTP activity and fibroblast activation. • Inorganic phosphate causes fibroblast activation independent of phospho-regulatory hormones.

  3. 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.

  4. 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.

  5. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

    International Nuclear Information System (INIS)

    Blanc-Brude, Olivier P.; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-01-01

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR 1 ). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR 1 -deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR 1 -specific agonists and inhibitors were used to demonstrate that PAR 1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR 1 and not PAR 2 . These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis

  6. 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.

  7. 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

  8. Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

    Science.gov (United States)

    Pérez, María J.; Ponce, Daniela P.; Osorio-Fuentealba, Cesar; Behrens, Maria I.; Quintanilla, Rodrigo A.

    2017-01-01

    The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients. PMID:29056898

  9. Antigen-presenting properties of gingival fibroblasts in chronic adult periodontitis

    NARCIS (Netherlands)

    Wassenaar, A.; Snijders, A.; Abraham-Inpijn, L.; Kapsenberg, M. L.; Kievits, F.

    1997-01-01

    Chronic periodontitis is characterized by dense infiltrations of T lymphocytes in the connective tissue, which consists mainly of gingival fibroblasts. It is becoming increasingly clear that T lymphocytes and gingival fibroblasts are capable of influencing each other. For example, the T cell

  10. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    Science.gov (United States)

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  11. Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

    International Nuclear Information System (INIS)

    Borek, C.

    1982-01-01

    In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

  12. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  13. Evidence of two distinct functionally specialized fibroblast lineages in breast stroma

    DEFF Research Database (Denmark)

    Morsing, Mikkel; Klitgaard, Marie Christine; Jafari Kermani, Abbas

    2016-01-01

    Background The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human...... conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271low/MUC1high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation...... fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial...

  14. Detection of genomic instability in α-irradiated and bystander human fibroblasts

    International Nuclear Information System (INIS)

    Ponnaiya, B.; Jenkins-Baker, G.; Bigelow, A.; Marino, S.; Geard, C.R.

    2003-01-01

    Full text: We have previously demonstrated a radiation induced bystander effect using novel co-culturing techniques where irradiated and bystander cells were cultured on two surfaces of mylar separated by media. Here we present data from experiments designed to investigate the induction of chromosomal aberrations in irradiated and bystander fibroblasts using these co-culturing techniques. Immortalized fibroblasts ((BJ1-tert) were cultured on both mylar surfaces and cells on one side were irradiated with 0.1 or 1 Gy -particles (an average of 1 and 10 particles per cell nucleus respectively), the two sides were separated 1 hour post irradiation and analyzed for chromosomal aberrations using standard Giemsa staining at either immediate or delayed time points. At 24-30 hours post irradiation, frequencies of chromosomal aberrations in irradiated populations were increased in a dose dependent manner as expected. Populations that received 0.1 and 1 Gy had 0.3 and 1.3 aberrations/cell; these aberrations were almost exclusively chromosome type aberrations. In contrast, at these times bystander populations had elevated yields of chromatid-type aberrations. When assayed at later times (15-20 population doublings post irradiation) both irradiated and bystander cells demonstrated elevated frequencies of chromatid-type aberrations. Frequencies in the irradiated populations ranged from 0.07 to 0.09 aberrations/ cell at 15 doublings, and 0.09-0.14/cell at 20 doublings, with no apparent dose response. Aberration frequencies in the bystander populations were between 0.08-0.14 per cell at the delayed time points assayed. Interestingly, the chromatid-type aberrations observed immediately post irradiation in the bystander cells, and at later times in both the irradiated and bystander populations were qualitatively similar to those previously observed at delayed times in neutron irradiated epithelial cells. Furthermore, there was a similar lack of a dose response in those studies as

  15. System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity.

    Directory of Open Access Journals (Sweden)

    Megha Rajaram

    Full Text Available Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8 or minimally (STC1 significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti

  16. 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.

  17. 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.

  18. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    International Nuclear Information System (INIS)

    Fan, Rong-hui; Zhu, Xiu-mei; Sun, Yao-wen; Peng, Hui-zi; Wu, Hang-li; Gao, Wen-jie

    2016-01-01

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  19. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Rong-hui, E-mail: fan_ronghuixa@163.com [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Zhu, Xiu-mei; Sun, Yao-wen [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Peng, Hui-zi [Department of Cosmetology Plastic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Wu, Hang-li; Gao, Wen-jie [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China)

    2016-07-08

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  20. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

  1. PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in fibroblasts from fish

    DEFF Research Database (Denmark)

    Ingerslev, Hans-Christian; Ossum, C.G.; Przybylska, Dominika

    . It is believed that this expression to a large extend is mediated by fibroblasts in the musculature. To investigate this, a fibroblast cell-line (RTHDF1) from the rainbow trout was stimulated with either LPS from E. coli, cell debris or supernatant from sonicated fibroblasts. Whereas LPS stimulation resulted...

  2. 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.)

  3. Lack of consent for mediation between companies and its reasons

    OpenAIRE

    Karpińska-Królikowska, Iwona

    2011-01-01

    This article discusses commercial mediation, presenting its principles and procedure. It shows the reason why I became interested in the topic of companies’ lack of willingness to solve problems through mediation. It presents empirical statistics from mediation in commercial cases, including those on lack of consents or settlements. The figures are shown against the background of court statistics. On the basis of research conducted in the form of case studies, it presents...

  4. Tattoo ink nanoparticles in skin tissue and fibroblasts.

    Science.gov (United States)

    Grant, Colin A; Twigg, Peter C; Baker, Richard; Tobin, Desmond J

    2015-01-01

    Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.

  5. Interleukin-1 inhibits the synthesis of collagen by fibroblasts.

    Science.gov (United States)

    Bhatnagar, R; Penfornis, H; Mauviel, A; Loyau, G; Saklatvala, J; Pujol, J P

    1986-10-01

    Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.

  6. Fibroblast Growth Factors: Biology, Function, and Application for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Ye-Rang Yun

    2010-01-01

    Full Text Available Fibroblast growth factors (FGFs that signal through FGF receptors (FGFRs regulate a broad spectrum of biological functions, including cellular proliferation, survival, migration, and differentiation. The FGF signal pathways are the RAS/MAP kinase pathway, PI3 kinase/AKT pathway, and PLCγ pathway, among which the RAS/MAP kinase pathway is known to be predominant. Several studies have recently implicated the in vitro biological functions of FGFs for tissue regeneration. However, to obtain optimal outcomes in vivo, it is important to enhance the half-life of FGFs and their biological stability. Future applications of FGFs are expected when the biological functions of FGFs are potentiated through the appropriate use of delivery systems and scaffolds. This review will introduce the biology and cellular functions of FGFs and deal with the biomaterials based delivery systems and their current applications for the regeneration of tissues, including skin, blood vessel, muscle, adipose, tendon/ligament, cartilage, bone, tooth, and nerve tissues.

  7. Response of human fibroblasts to low dose rate gamma irradiation

    International Nuclear Information System (INIS)

    Dritschilo, A.; Brennan, T.; Weichselbaum, R.R.; Mossman, K.L.

    1984-01-01

    Cells from 11 human strains, including fibroblasts from patients with the genetic diseases of ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Fanconi's anemia (FA), were exposed to γ radiation at high (1.6-2.2 Gy/min) and at low (0.03-0.07 Gy/min) dose rates. Survival curves reveal an increase inthe terminal slope (D 0 ) when cells are irradiated at low dose rates compared to high dose rates. This was true for all cell lines tested, although the AT, FA, and XP cells are reported or postulated to have radiation repair deficiencies. From the response of these cells, it is apparent that radiation sensitivities differ; however, at low dose rate, all tested human cells are able to repair injury

  8. Induction of pluripotent stem cells from fibroblast cultures.

    Science.gov (United States)

    Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

    2007-01-01

    Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

  9. 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.

  10. The dependence of fibroblast radiosensitivity on cell pH

    International Nuclear Information System (INIS)

    Veksler, A.M.; Kublik, L.N.; Degtyareva, O.V.; Ehjdus, L.Kh.

    1983-01-01

    The problem of the change of radiosensitivity of Chinese hamster fibroblasts, irradiated under aerobic and hypoxic conditions in the course of intracellular pH (pHsub(intr.)) change by means of a phosphate buffer has been studied. It has been found that pHsub(intr.) reduction considerably increases the radiosensitivity, the effect being more pronounced on hypoxic cells which is essential for radiotherapy of tumors. The survival rate of cell irradiated under hypoxia conditions does not depend on season while cell resistance in case of irradiation in open air in spring and autumn is different. The effect discovery in case of pHsub(intr.) reduction upon irradiation shows up the influence of the studied factor on repair processes

  11. Malignant transformation of diploid human fibroblasts by transfection of oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, J.J.

    1992-01-01

    This document consist of brief reports prepared by postdoctoral students supported by the project, each describing his accomplishments under the grant. Topics include (1) Malignant Transformation of MSU-1. 1 Cells by Gamma Radiation, (2) Correlation between Levels of ras Expression and Presence of Transformed Phenotypes Including Tumorigenicity, Using a Modulatable Promoter, (3) Relation between Specific rad Oncogene Expression, (4) Correlation of Genetic Changes in Fibroblastic Tumors with Malignancies, (5)Transformation of MSU-1.1 Cells by sis Oncogene, (6) Malignant Transformation of MSU-1.0 Cells, (7) Correlation of Urokinase Plasminogen Activation (mu-PA) with Malignant Phenotype, (8)Two Dimensional Gel Electrophoresis Studies of the Proteins of the Major Cell Strains of the MSU-1 Family of Cells, and (9) Correlation between Proteinase Activity Levels and Malignancy.

  12. Tattoo ink nanoparticles in skin tissue and fibroblasts

    Directory of Open Access Journals (Sweden)

    Colin A. Grant

    2015-05-01

    Full Text Available Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.

  13. Acetylome in Human Fibroblasts From Parkinson's Disease Patients

    Directory of Open Access Journals (Sweden)

    Sokhna M. S. Yakhine-Diop

    2018-04-01

    Full Text Available Parkinson's disease (PD is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2 are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

  14. DNA single strand break in fibroblast from Down syndrome patients

    International Nuclear Information System (INIS)

    Rozga, B.

    1992-01-01

    The radiosensitivity of tree trisomic (trisomia +21) strains of human fibroblasts to gamma radiation has been investigated in vitro and the causes of induction and repair of single strand DNA breaks in these cells have been estimated. The single strand breaks in DNA of normal and trisomic cells have been found to be ameliorated with an approximately equal efficiency. Repair has been found to be three times slower in trisomic cells compared to their normal relevant, most likely due to their elevated sensitivity to ionizing radiation and the following mortality of trisomic cells, and/or the potential occurrence of a great number of chromosome aberrations in cells irradiated in vitro. (author). 28 refs, 4 figs, 1 tab

  15. Fibroblast Growth Factor 23 in Long-Duration Spaceflight

    Science.gov (United States)

    Bokhari, R.; Zwart, S. R.; Fields, E.; Heer, M.; Sibonga, J.; Smith, S. M.

    2015-01-01

    Many nutritional factors influence bone, from the basics of calcium and vitamin D, to factors which influence bone through acid/base balance, including protein, sodium, and more. Fibroblast growth factor 23 (FGF23) is a recently identified factor, secreted from osteocytes, which is involved in classic (albeit complex) feedback loops controlling phosphorus homeostasis through both vitamin D and parathyroid hormone (PTH) (1, 2). As osteocytes are gravity sensing cells, it is important to determine if there are changes in FGF23 during spaceflight. In extreme cases, such as chronic kidney disease, FGF23 levels are highly elevated. FGF23 imbalances, secondary to dietary influences, may contribute to skeletal demineralization and kidney stone risk during spaceflight.

  16. Malignant transformation of diploid human fibroblasts by transfection of oncogenes

    International Nuclear Information System (INIS)

    McCormick, J.J.

    1992-01-01

    This document consist of brief reports prepared by postdoctoral students supported by the project, each describing his accomplishments under the grant. Topics include (1) Malignant Transformation of MSU-1. 1 Cells by Gamma Radiation, (2) Correlation between Levels of ras Expression and Presence of Transformed Phenotypes Including Tumorigenicity, Using a Modulatable Promoter, (3) Relation between Specific rad Oncogene Expression, (4) Correlation of Genetic Changes in Fibroblastic Tumors with Malignancies, (5)Transformation of MSU-1.1 Cells by sis Oncogene, (6) Malignant Transformation of MSU-1.0 Cells, (7) Correlation of Urokinase Plasminogen Activation (mu-PA) with Malignant Phenotype, (8)Two Dimensional Gel Electrophoresis Studies of the Proteins of the Major Cell Strains of the MSU-1 Family of Cells, and (9) Correlation between Proteinase Activity Levels and Malignancy

  17. C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.

    Science.gov (United States)

    Kimura, Toru; Nojiri, Takashi; Hino, Jun; Hosoda, Hiroshi; Miura, Koichi; Shintani, Yasushi; Inoue, Masayoshi; Zenitani, Masahiro; Takabatake, Hiroyuki; Miyazato, Mikiya; Okumura, Meinoshin; Kangawa, Kenji

    2016-02-19

    Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22

  18. Tumor-produced, active Interleukin-1 β regulates gene expression in carcinoma-associated fibroblasts

    International Nuclear Information System (INIS)

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-01-01

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-β. PDL fibroblasts possess receptor for IL1-β, and its expression is increased 4.56-times in the presence of SCC-25 tumor cells. IL1-β receptor expression in

  19. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  20. LXA{sub 4} actions direct fibroblast function and wound closure

    Energy Technology Data Exchange (ETDEWEB)

    Herrera, Bruno S. [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Leung, Kai P., E-mail: kai.p.leung.civ@mail.mil [Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Van Dyke, Thomas E., E-mail: tvandyke@forsyth.org [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States)

    2015-09-04

    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{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 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{sub 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{sub 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{sub 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{sub 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{sub 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

  1. Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

    Science.gov (United States)

    Mackey, Abigail L; Magnan, Mélanie; Chazaud, Bénédicte; Kjaer, Michael

    2017-08-01

    Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable

  2. Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

    2011-11-01

    The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Effects of plant lectins on in vitro fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Ana Maria Sell

    2003-06-01

    Full Text Available Lectins are carbohydrate-binding proteins that have been isolated from various sources and presented a wide spectrum of biological activities. The effects of four lectins, namely, Phaseolus vulgaris phytohemagglutinin, PHA, wheat germ agglutinin, WGA, Artocarpus integrifolia seed lectins, jacalin and artocarpin, on in vitro fibroblasts proliferation were investigated. The lectins did not influence the initial cell adhesion to the plate. PHA and WGA at 10-20 µg/mL concentrations significantly decreased fibroblasts proliferation. At these concentrations, they caused morphological alterations on cells and over 80 µg/mL, promoted cell death. Neither jacalin nor artocarpin significantly affected cell proliferation.O objetivo deste trabalho foi avaliar a influência das lectinas PHA, WGA, jacalina e artocarpina sobre a proliferação de fibroblastos in vitro. Para tanto, fibroblastos gengivais de voluntários saudáveis foram cultivados, por cinco dias, em DMEM suplementado com soro bovino fetal (10% v/v e na presença das lectinas nas concentrações finais de 0.1 a 300 µg/mL. A adesão, o crescimento e a morfologia celular foram acompanhados por microscopia de inversão e contraste de fase. O índice de proliferação foi avaliado pelo método calorimétrico usando MTT. As lectinas não alteraram a adesão inicial dos fibroblastos à placa de poliestireno. PHA e WGA, nas concentrações de 10 a 20 µg/mL, diminuíram significativamente a proliferação celular. Nestas concentrações a morfologia celular é alterada e acima de 80 µg/mL, houve100% de morte celular. As lectinas jacalina e artocarpina não influenciaram a proliferação celular.

  4. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  5. Concentration- and time-dependent response of human gingival fibroblasts to fibroblast growth factor 2 immobilized on titanium dental implants

    Directory of Open Access Journals (Sweden)

    Ma Q

    2012-04-01

    Full Text Available Qianli Ma1*, Wei Wang1*, Paul K Chu2, Shenglin Mei1,2, Kun Ji3, Lei Jin4, Yumei Zhang11Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China; 2Department of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong Kong, People's Republic of China; 3Department of Pediatric Dentistry, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China; 4Stomatology Department, Jinling Hospital, School of Medicine, Southern Medical University, Nanjing, People's Republic of China*These authors contributed equally to this workBackground: Titanium (Ti implants are widely used clinically, but peri-implantitis remains one of the most common and serious complications. Healthy integration between gingival tissue and the implant surface is critical to long-term success in dental implant therapy. The objective of this study was to investigate how different concentrations of immobilized fibroblast growth factor 2 (FGF2 on the titania nanotubular surface influence the response of human gingival fibroblasts (HGFs.Methods: Pure Ti metal was anodized at 20 V to form a vertically organized titanium dioxide nanotube array on which three concentrations of FGF2 (250 ng/mL, 500 ng/mL, or 1000 ng/mL were immobilized by repeated lyophilization. Surface topography was observed and FGF2 elution was detected using enzyme-linked immunosorbent assay. The bioactivity changes of dissolvable immobilized FGF2 were measured by methyl-thiazolyl-tetrazolium assay. Behavior of HGFs was evaluated using adhesion and methyl-thiazolyl-tetrazolium bromide assays.Results: The FGF2 remained for several days on the modified surface on which HGFs were cultured. Over 90% of the dissolvable immobilized FGF2 had been eluted by Day 9, whereas the FGF2 activity was found to diminish gradually from Day 1 to Day 9. The titania nanotubular surface with an optimal preparing

  6. Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

    Science.gov (United States)

    Janson, David; Rietveld, Marion; Mahé, Christian; Saintigny, Gaëlle; El Ghalbzouri, Abdoelwaheb

    2017-06-01

    Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

  7. Identification of specific gene expression profiles in fibroblasts derived from middle ear cholesteatoma.

    Science.gov (United States)

    Yoshikawa, Mamoru; Kojima, Hiromi; Wada, Kota; Tsukidate, Toshiharu; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

    2006-07-01

    To investigate the role of fibroblasts in the pathogenesis of cholesteatoma. Tissue specimens were obtained from our patients. Middle ear cholesteatoma-derived fibroblasts (MECFs) and postauricular skin-derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1alpha and/or IL-1beta before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs. Six patients who had undergone tympanoplasty. The IL-1alpha-regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC (liver and activation-regulated chemokine), GMCSF (granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1 (intercellular adhesion molecule 1), and TGFA (transforming growth factor alpha) were more strongly up-regulated by IL-1alpha and/or IL-1beta in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles. Fibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.

  8. Effect of eosinophils activated with Alternaria on the production of extracellular matrix from nasal fibroblasts.

    Science.gov (United States)

    Shin, Seung-Heon; Ye, Mi-Kyung; Choi, Sung-Yong; Kim, Yee-Hyuk

    2016-06-01

    Eosinophils and fibroblasts are known to play major roles in the pathogenesis of nasal polyps. Fungi are commonly found in nasal secretion and are associated with airway inflammation. To investigate whether activated eosinophils by airborne fungi can influence the production of extracellular matrix (ECM) from nasal fibroblasts. Inferior turbinate and nasal polyp fibroblasts were stimulated with Alternaria or Aspergillus, respectively, for 24 hours and ECM messenger RNA (mRNA) and protein expressions were measured. Eosinophils isolated from healthy volunteers were stimulated with Alternaria or Aspergillus for 4 hours then superoxide, eosinophil peroxidase, and transforming growth factor β1 were measured. Then activated eosinophils were cocultured with nasal fibroblasts for 24 hours, and ECM mRNA expressions were measured. Alternaria strongly enhanced ECM mRNA expression and protein production from nasal fibroblasts. Alternaria also induced the production of superoxide, eosinophil peroxidase, and transforming growth factor β1 from eosinophils, and activated eosinophils enhanced ECM mRNA expression when they were cocultured without the Transwell insert system. Eosinophils activated with Alternaria enhanced ECM mRNA expression from nasal polyp fibroblasts. Alternaria plays an important role in tissue fibrosis in the pathogenesis of nasal polyps by directly or indirectly influencing the production of ECM from nasal fibroblasts. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

    Directory of Open Access Journals (Sweden)

    Dongyuan Lü

    Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

  10. Fibroblast growth factor receptor 4 regulates tumor invasion by coupling fibroblast growth factor signaling to extracellular matrix degradation

    DEFF Research Database (Denmark)

    Sugiyama, Nami; Varjosalo, Markku; Meller, Pipsa

    2010-01-01

    /stroma border and tumor invasion front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMP-dependent collagen invasion......Aberrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to tumor progression and anticancer drug resistance. We describe here a novel mechanism of tumor progression by matrix degradation involving epithelial-to-mesenchymal transition in response...... to membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas, where they were partially coexpressed at the tumor...

  11. Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

    Science.gov (United States)

    Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

    2017-11-01

    Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

  12. 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.

  13. Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

    Directory of Open Access Journals (Sweden)

    Allison L Speer

    Full Text Available The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10 and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b, in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22 except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

  14. Induction of growth stimulation in skin fibroblasts from retinoblastoma donors after ionizing radiation

    International Nuclear Information System (INIS)

    Diatloff-Zito, C.; Macieira-Coelho, A.; Turleau, C.; Cabanis, M.O.; Grouchy, J. de

    1983-01-01

    Skin fibroblasts from normal children and two children with a 13q14 deletion retinoblastoma (Rb) were submitted to fractionated doses of γ radiations. Irradiation reduced the population doublings in normal fibroblasts and the decline was inversely related to the dose. An increase in population doublings was obtained with one of the Rb cell lines. Foci appeared in the irradiated culture of the other Rb donor. It is suggested that fibroblasts from patients with Rb are able to express some phenotypical properties of transformed cells, perhaps related to factors rendering them more susceptible to carcinogens [fr

  15. 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

  16. Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA

    International Nuclear Information System (INIS)

    Day, R.S. III; Kraemer, K.H.; Robbins, J.H.

    1975-01-01

    UV survival curves of adenovirus 2 using fused complementing xeroderma pigmentosum fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusions involved strains in the same complementing group. Extrapolation to zero dose indicated that three percent of the viral plaque-forming units had infected cells capable of normal repair; this suggested that three percent of the cells were complementing heterokaryons. Thus, heterokaryons formed from xeroderma pigmentosum fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells

  17. Human fibroblast strain with normal survival but abnormal postreplication repair after ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Doniger, J.; Barrett, S.F.; Robbins, J.H.

    1980-01-01

    Postreplication repair has been studied in ultraviolet light (UV-irradiated) fibroblast strains derived from eight apparently normal control donors and seven xeroderma pigmentosum patients. One control donor strain had an intermediate defect in postreplication repair similar to that in excision-deficient xeroderma pigmentosum fibroblasts. However, unlike the xeroderma pigmentosum strains, this control donor strain had normal UV-induced unscheduled DNA synthesis and normal survival after irradiation with UV. This unique fibroblast strain should be useful in studies designed to elucidate the possible role of postreplication repair in UV-induced carcinogenesis and mutagenesis

  18. Lack of dust in quasar absorption line systems

    International Nuclear Information System (INIS)

    Jura, M.

    1977-01-01

    It is stated that the origin of absorption line systems in quasars is still uncertain. Most such systems apparently have atomic hydrogen column densities of the order of 10 19 /cm 2 , but at least two quasars, 1331 + 170 and PHL957, have such strong Lyman α absorption lines that atomic hydrogen column densities of the order of 10 21 /cm 2 are indicated. It should be possible to observe the dust produced 2,200 A extinction feature as it is red shifted into the visible, and to determine whether absorption line systems are produced in spiral galaxies where the dust content is similar to that in the interstellar medium. It has been argued that the emission line regions of quasars generally lack dust and that towards PHL957 the 2,200 A feature is absent. The present author argues that dust similar to that found in the interstellar medium is not found towards the quasars 1331 + 170 and PHL957. This could explain why H 2 is not found towards PHL957, and it indicates that the absorption line systems in quasars are not produced in spiral galaxies similar to our own. It seems from the analysis presented that the dust-to-gas ratio towards 1331 + 170 is at least a factor of 20 less than in the interstellar medium, and there is no reason to suppose that this lack of dust results from a lack of metals It is concluded that there seems to be a lack of normal dust towards PHL957 by at least a factor of two; and that the absorption region towards 1331 + 170 and probably the region towards PHL957 are lacking dust similar to that in our own galaxy. This can explain the lack of H 2 in these systems. (U.K.)

  19. Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

    International Nuclear Information System (INIS)

    Gery, Bernard; Little, John B.; Coppey, Jacques

    1996-01-01

    Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. Methods and Materials: The cell system consists of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. Results: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10 5 fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. Conclusions: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumors fragments

  20. Effect of lack of later support in the masseter muscle

    International Nuclear Information System (INIS)

    Fernandez Lopez, Otton

    2007-01-01

    One of the main complaints during dental consultation has been pain in the zone of the masseter muscle, especially a lack of rear support. None research has published that reveals what has been the relationship between the rear support and histological alterations in muscle mass. Both topics have treated to relate through a process of tooth wear in laboratory animals and produce a lack of rear support. Cuts of the masseter muscles and specimens were subjected to microscopic study of light and electronic. The conclusion has been that by removing the rear support are produced important changes to histological level. (author) [es

  1. [Lack of neonatologists: vocational crisis or mistaken policies?].

    Science.gov (United States)

    Justich, Pablo R

    2012-10-01

    In Argentina, the difficulty in covering neonatologist's positions represent an increasing problem. The absence of a coordinated and organized health system on one hand, and the lack of adaptation of the neonatologist's role to the current situation of the maternal and child care on the other, prevent the correct assistential coverage. The inadequate work conditions, the professional risks, the wide amount of time devoted to formation and studying, and the lack of knowledge of the professionals necessities and difficulties have a negative impact when it comes to incorporate new specialists. A global approach of the problem is essential to reach enduring answers.

  2. Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Rachel C Williams

    Full Text Available Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival

  3. Cytotoxic Effects of Nickel Nanowires in Human Fibroblasts

    KAUST Repository

    Felix Servin, Laura P.

    2014-04-01

    There is an increasing interest for the use of nanostructures as potential tools in areas that include biology and medicine, for applications spanning from cell separation to treatments of diseases. Magnetic nanoparticles (MNPs) have been the most widely studied and utilized nanostructures in biomedical applications. Despite their popularity, the regular shape of MNPs limits their potential for certain applications. Studies have shown that magnetic nanowires (MNWs), due to their high-­‐aspect ratio and specific magnetic properties, might provide improved performance for some biomedical applications. As a consequence, MNWs have received increasing attention from researchers in the last years. However, as with any other nanostructure intended for biomedical applications, rigorous studies must be carried out to determine their potential toxicity and adverse effects before they can be successfully incorporated in clinical applications. This work attempts to elucidate the cytotoxic effects of nickel NWs (Ni NWs) in human fibroblasts by measuring cell viability under different parameters. Ni NWs of three different lengths (0.86 ± 0.02 μm, 1.1 ± 0.1 μm and 6.1 ± 0.6 μm) were fabricated by electrodeposition using porous aluminum oxide (PAO) membranes as templates. Energy dispersive X-­‐Ray analysis (EDAX) and X-­‐Ray diffraction (XRD) were used for the chemical characterization of the Ni NWs. Their physical characterization was done using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging. MTT assays were performed to assess cell viability of human fibroblasts in the presence of Ni NWs. NW length, NW/cell ratio and exposure time were changed throughout the experiments to elucidate their effects on cell viability. The results showed that NWs length has a strong effect on internalization and cytotoxicity. Smaller NWs showed higher toxicity levels at earlier times while longer NWs had stronger effects on cell viability at

  4. Cytotoxic Effects of Nickel Nanowires in Human Fibroblasts

    KAUST Repository

    Felix Servin, Laura P.

    2014-01-01

    There is an increasing interest for the use of nanostructures as potential tools in areas that include biology and medicine, for applications spanning from cell separation to treatments of diseases. Magnetic nanoparticles (MNPs) have been the most widely studied and utilized nanostructures in biomedical applications. Despite their popularity, the regular shape of MNPs limits their potential for certain applications. Studies have shown that magnetic nanowires (MNWs), due to their high-­‐aspect ratio and specific magnetic properties, might provide improved performance for some biomedical applications. As a consequence, MNWs have received increasing attention from researchers in the last years. However, as with any other nanostructure intended for biomedical applications, rigorous studies must be carried out to determine their potential toxicity and adverse effects before they can be successfully incorporated in clinical applications. This work attempts to elucidate the cytotoxic effects of nickel NWs (Ni NWs) in human fibroblasts by measuring cell viability under different parameters. Ni NWs of three different lengths (0.86 ± 0.02 μm, 1.1 ± 0.1 μm and 6.1 ± 0.6 μm) were fabricated by electrodeposition using porous aluminum oxide (PAO) membranes as templates. Energy dispersive X-­‐Ray analysis (EDAX) and X-­‐Ray diffraction (XRD) were used for the chemical characterization of the Ni NWs. Their physical characterization was done using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging. MTT assays were performed to assess cell viability of human fibroblasts in the presence of Ni NWs. NW length, NW/cell ratio and exposure time were changed throughout the experiments to elucidate their effects on cell viability. The results showed that NWs length has a strong effect on internalization and cytotoxicity. Smaller NWs showed higher toxicity levels at earlier times while longer NWs had stronger effects on cell viability at

  5. Children's Lack of Playtime Seen as Troubling Health, School Issue

    Science.gov (United States)

    Jacobson, Linda

    2008-01-01

    Teachers and parents are frequently warned that students in the United States are lacking the academic skills they need for the 21st century. But a growing contingent of educators, psychologists, and other professionals are voicing worries that today's children are also growing up without the chance to play. Test preparation in kindergarten,…

  6. Lack of competition in Italian natural gas market

    International Nuclear Information System (INIS)

    Bozzetto, Fabrizio

    2007-01-01

    This article analyses the reasons for an evident lack of competition in the Italian natural gas market, after the 2003 full liberalisation of the market. In particular, analysis focuses on dynamics which probably marks mass market and small office segments [it

  7. Kidney failure in mice lacking the tetraspanin CD151

    NARCIS (Netherlands)

    Sachs, Norman; Kreft, Maaike; van den Bergh Weerman, Marius A.; Beynon, Andy J.; Peters, Theo A.; Weening, Jan J.; Sonnenberg, Arnoud

    2006-01-01

    The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin alpha3beta1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking

  8. Kidney failure in mice lacking the tetraspanin CD151.

    NARCIS (Netherlands)

    Sachs, N.; Kreft, M.; Bergh Weerman, M. van der; Beynon, A.J.; Peters, T.A.; Weening, J.J.; Sonnenberg, A.

    2006-01-01

    The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin alpha3beta1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking

  9. Siim Nestor soovitab : lack of Eoins / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2008-01-01

    Väikefirma Seksound annab sel nädalavahetusel välja Viljandi indiebändi Lack of Eoins esikplaadi "Echo Group" (plaadiesitlused 11. dets. Tallinnas Von Krahlis ja 12. dets. Tartus Genialistide klubis, esinevad ka Ans. Andur ja Popidiot, plaate keerutavad Hannes Praks ja Taavi Laatsit)

  10. Lack of a safety culture destroyed the reactor

    International Nuclear Information System (INIS)

    Vuori, A.

    1996-01-01

    The importance of good safety culture in the operation of nuclear power plants is discussed. The modern safety culture emphasizes responsibility and preventive maintenance that can eliminate or minimize faults in advance. In the article the accident of Chernobyl is used as an example of the lack of safety culture. (1 fig.)

  11. Special Relativity in Week One: 4) Lack of Simultaneity

    Science.gov (United States)

    Huggins, Elisha

    2011-01-01

    This is our final article on teaching special relativity in the first week of an introductory physics course. One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the…

  12. Lack of pre-antiretroviral care and competition from traditional ...

    African Journals Online (AJOL)

    Lack of family support tripled the risk of initiating ART very late (AOR 3.3, 95% CI: 1.6-6.6). Conclusion: Policy makers should prevent ARV stock-outs though effective ARV procurement and supply chain management. New HIV clients should seek pre-ARV care for routine monitoring and determination of ART eligibility.

  13. Alpha8 Integrin (Itga8 Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover.

    Directory of Open Access Journals (Sweden)

    Ines Marek

    Full Text Available The α8 integrin (Itga8 chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration.

  14. Photobiomodulation changes type 1 collagen gene expression by pulp fibroblasts

    Science.gov (United States)

    Lourenço Ribeiro Vitor, Luciana; Tavares Oliveira Prado, Mariel; Lourenço Neto, Natalino; Cardoso de Oliveira, Rodrigo; Ferreira Santos, Carlos; Moreira Machado, Maria Aparecida Andrade; Marchini Oliveira, Thais

    2018-06-01

    This study aimed to evaluate type 1 collagen (COL1) gene expression by human pulp fibroblasts from primary teeth (HPF) after the variation of photobiomodulation (PBM) parameters. HPF were obtained from a biorepository, used at 4th passage, and irradiated (InGaAlP—660 nm) varying the power and application time according to the following groups: G1: 1.2 J cm‑2–05 mW–10 s G2: 2.5 J cm‑2–05 mW–20 s G3: 3.7 J cm‑2–05 mW–30 s G4: 5.0 J cm‑2–05 mW–40 s G5: 6.2 J cm‑2–05 mW–50 s G6: 2.5 J cm‑2–10 mW–10 s G7: 3.7 J cm‑2–15 mW–10 s G8: 5.0 J cm‑2–20 mW–10 s G9: 6.2 J cm‑2–25 mW–10 s. The control group (G10) was not irradiated and maintained with DMEM  +  10% SFB. RT-PCR was used to evaluate COL1 gene expression at 6, 12, and 24 h after irradiation. Intra- and intergroup comparisons were performed by two-way ANOVA followed by Tukey test (p  differences among periods (p  differences (p  >  0.05). The energy densities from 2.5 to 5 J cm‑2, regardless of the variation in PBM parameters, biomodulated the COL1 gene expression. At the energy density of 6.2 J cm‑2, longer application time and smaller power changed the pattern of COL1 gene expression by pulp fibroblasts from human primary teeth.

  15. Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

    Science.gov (United States)

    Gunnink, Stephen M; Kerk, Samuel A; Kuiper, Benjamin D; Alabi, Ola D; Kuipers, David P; Praamsma, Riemer C; Wrobel, Kathryn E; Louters, Larry L

    2014-04-01

    The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  16. Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

    Science.gov (United States)

    Wu, Yuanyuan; Wang, Karen; Karapetyan, Adrine; Fernando, Warnakulusuriya Akash; Simkin, Jennifer; Han, Manjong; Rugg, Elizabeth L.; Muneoka, Ken

    2013-01-01

    A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue

  17. Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Wu

    Full Text Available A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3 leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2, fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3 and incompetent (P2 levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of

  18. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Leah J.; Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Kandpal, Sanjeev Kumar; Mason, Michael D. [Department of Chemical and Biological Engineering, University of Maine, Orono, ME (United States); Zheng, Tongzhang [Department of Environmental Health Sciences, Yale School of Public Health, New Haven, CT (United States); Wise, John Pierce, E-mail: John.Wise@usm.maine.edu [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States)

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. - Highlights: • Particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells. • Soluble cobalt induces more cytotoxicity compared to particulate cobalt. • Soluble and particulate cobalt induce similar levels of genotoxicity. • Particle-cell contact is required for particulate cobalt-induced toxicity.

  19. SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

    Science.gov (United States)

    Stein, G H

    1985-10-01

    Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

  20. SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

    Science.gov (United States)

    Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

    1990-01-01

    To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

  1. Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

    Science.gov (United States)

    Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

    2018-03-05

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

    International Nuclear Information System (INIS)

    Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I.

    1989-01-01

    Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125 I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10 12 binding sites/mm 2 ECM with an apparent k D of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 μg/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 μg/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response

  3. Inhibition of proliferation and migration of stricture fibroblasts by epithelial cell-conditioned media

    Directory of Open Access Journals (Sweden)

    Nilima Nath

    2015-01-01

    Conclusion: These results demonstrate the ability of ECCM to inhibit the proliferation and migration of stricture fibroblasts and present it as an effective adjunct in urethroplasty, which may influence stricture wound healing and inhibit the recurrence of stricture.

  4. Receptor protein tyrosine phosphatase alpha enhances rheumatoid synovial fibroblast signaling and promotes arthritis in mice

    NARCIS (Netherlands)

    Stanford, Stephanie M; Svensson, Mattias N D; Sacchetti, Cristiano; Pilo, Caila A; Wu, Dennis J; Kiosses, William B; Hellvard, Annelie; Bergum, Brith; Aleman Muench, German R; Elly, Christian; Liu, Yun-Cai; den Hertog, Jeroen; Elson, Ari; Sap, Jan; Mydel, Piotr; Boyle, David L; Corr, Maripat; Firestein, Gary S; Bottini, Nunzio

    2016-01-01

    OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the joint extracellular matrix. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to RA FLS anomalous behavior. The receptor

  5. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    National Research Council Canada - National Science Library

    Chang, Howard Y

    2008-01-01

    The results from year I of funding suggest that appropriate markers and technologies are in place for systematic investigation of the positional identity of fibroblasts both in normal and diseased tissues...

  6. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    International Nuclear Information System (INIS)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

    1990-01-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

  7. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena; Carbone, Ennio; Di Fabrizio, Enzo M.; Tirinato, Luca; Petruchek, Maria; Drummond, Catherine; Kovalevska, Larysa; Gurrapu, Sreeharsha; Mushtaq, Muhammad; Darekar, Suhas D.

    2015-01-01

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways

  8. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    Science.gov (United States)

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. © The Author(s) 2016. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.

  9. Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors.

    Science.gov (United States)

    Figueroa, R; Lindenmaier, H; Hergenhahn, M; Nielsen, K V; Boukamp, P

    2000-06-01

    The life span of normal fibroblasts in vitro (Hayflick limit) depends on donor age, and telomere shortening has been proposed as a potential mechanism. By quantitative fluorescence in situ hybridization and Southern blot analysis, we show progressive telomere loss to about 5 kb mean telomere restriction fragment length in fibroblasts from two adult donors within 40 population doublings, whereas in fibroblasts from two infant donors, telomere erosion is reduced, leaving a mean telomere restriction fragment length of approximately 7 kb at senescence (after approximately 60 population doublings). Aging of fibroblasts from both infant and adult donors was not accompanied by chromosomal abnormalities but was correlated with increased telomere repeat-binding factor 2 expression at both the protein and transcriptional level.

  10. Clearance of Sclerostin, Osteocalcin, Fibroblast Growth Factor 23, and Osteoprotegerin by Dialysis

    DEFF Research Database (Denmark)

    Carlson, Nicholas; Mortensen, Ole H.; Axelsen, Mette

    2017-01-01

    Introduction: Fibroblast growth factor (FGF23), sclerostin, osteocalcin, and osteoprotegerin are important factors that control mineral bone metabolism. End-stage renal disease is associated with the pronounced dysregulation of mineral bone metabolism; however, the impact and clearance of mineral...

  11. DNA repair in Bloom's syndrome skin fibroblasts after ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Kurihara, Takayuki; Inoue, Masao; Kawashima, Hiroko; Yagi, Takashi; Takebe, Hiraku.

    1981-01-01

    Skin fibroblasts from a patient with Bloom's syndrome (86NoKi) were assayed for various DNA repair activities after ultraviolet light (UV) irradiation. Cultured fibroblasts as well as lymphocytes obtained from this patient showed a high frequency of spontaneous sister chromatid exchanges (SCEs). There was no significant difference between 86NoKi fibroblasts and skin fibroblasts from normal donors in the sensitivity to UV as measured by inactivation of colony forming activity, the capacity of host-cell reactivation (HCR) of UV-irradiated virus, and the amount of unscheduled DNA synthesis (UDS) after UV irradiation. However, the yield of UV-induced SCEs in 86NoKi cells was significantly higher than that in normal cells. (author)

  12. Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

    Directory of Open Access Journals (Sweden)

    Cheng M

    2014-12-01

    Full Text Available Michelle Cheng,1,* Samantha Ho,1,* Jun Hwan Yoo,1,2,* Deanna Hoang-Yen Tran,1,* Kyriaki Bakirtzi,1 Bowei Su,1 Diana Hoang-Ngoc Tran,1 Yuzu Kubota,1 Ryan Ichikawa,1 Hon Wai Koon1 1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA; 2Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Republic of Korea *These authors share co-first authorship Background: Cathelicidin (LL-37 in humans and mCRAMP in mice represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the

  13. Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

    Directory of Open Access Journals (Sweden)

    L. V. Altukhova

    2015-09-01

    Full Text Available Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160×103 fibroblasts and (130–140×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in

  14. Repair of ultraviolet light damage to the DNA of cultured human epidermal keratinocytes and fibroblasts

    International Nuclear Information System (INIS)

    Taichman, L.B.; Setlow, R.B.

    1979-01-01

    Pure cultures of dermal fibroblasts and epidermal keroatinocytes have been obtained from a single biopsy of newborn foreskin. The cells were labeled, exposed to several doses of uv light, and allowed to repair in the dark for 16 h. The number of pyrimidine dimers before and after repair was assessed by measuring the numbers of sites in the DNA sensitive to a specific uv endonuclease. At all doses used, the extent of repair was similar in the cultured keratinocytes and cultured fibroblasts

  15. Role of postreplication repair in transformation of human fibroblasts to anchorage independence

    International Nuclear Information System (INIS)

    Boyer, J.C.; Kaufmann, W.K.; Cordeiro-Stone, M.

    1991-01-01

    Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m 2 and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m 2 and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m 2 ; XP variant fibroblasts, 0.3 J/m 2 ) and BPDE-I (both, 0.05 microM)

  16. Constituents from the roots of Taraxacum platycarpum and their effect on proliferation of human skin fibroblasts.

    Science.gov (United States)

    Warashina, Tsutomu; Umehara, Kaoru; Miyase, Toshio

    2012-01-01

    A MeOH extract from the roots of Taraxacum platycarpum has shown significant effects on the proliferation of normal human skin fibroblasts. Chemical analysis of the extract resulted in the isolation of 26 compounds, including eight new triterpenes, one new sesquiterpene glycoside, and seventeen known compounds. The structure of each new compound was established using NMR spectroscopy. Some triterpenes had a significant effect on the proliferation of normal human skin fibroblasts.

  17. Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

    Science.gov (United States)

    Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

    2015-01-01

    Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

  18. Proliferative and inductive effects of Cyclosporine a on gingival fibroblast of child and adult

    Directory of Open Access Journals (Sweden)

    Bahareh Nazemi Salman

    2013-01-01

    Conclusions: The mechanism of a CsA-induced fibroblast overgrowth may converge on the steps involving fibroblast proliferation and cytokine network including IL-6, IL-8, IL-1β, TGF-β1, and PGE 2 , in both adults and pediatrics. As the prevalence and intensity of drug-induced gingival overgrowth is more serious in the pediatrics. As group than in adults, we suggest that more studies be conducted on the pediatric group.

  19. Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi's anemia

    International Nuclear Information System (INIS)

    Remsen, J.F.; Cerutti, P.A.

    1976-01-01

    The capacity of preparations of skin fibroblasts from normal individuals and patients with Fanconi's anemia to excise gamma-ray products of the 5,6-dihydroxydihydrothymine type from exogenous DNA was investigated. The excision capacity of whole-cell homogenates of fibroblasts from two of four patients with Fanconi's anemia was substantially below normal. This repair deficiency was further pronounced in nuclear preparations from cells of the same two patients

  20. Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

    Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

  1. Generation of hiPSTZ16 (ISMMSi003-A cell line from normal human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    Marion Dejosez

    2018-01-01

    Full Text Available Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A. These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

  2. Effect of glutathione on arecanut treated normal human buccal fibroblast culture.

    Directory of Open Access Journals (Sweden)

    Saraswathi T

    2006-01-01

    Full Text Available BACKGROUND: Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential. OBJECTIVE: The present study was undertaken to evaluate the effect of glutathione on arecanut treated human buccal fibroblast culture and its potential as a chemopreventive agent. MATERIALS AND METHODS: Fibroblast culture was done in Dulbecco′s Modified Eagle′s Medium MEM supplemented with 10% Fetal Calf Serum (FCS and antibiotic at 370C degrees in an atmosphere of 5% carbon di-oxide and 95% air. The fibroblast cells were subjected to different concentrations of aqueous extracts of raw and boiled arecanut. Fibroblasts were plated in two 24-well culture plates and in each plate, cells were dividt,ednto 2 groups; 600gg microml of reduced glutathione was added to the first group of cells; subsequently, aqueous extracts of raw and boiled arecanut at least and highest concentrations i.e., 20j. microml and 100lg microml were added to the first group of cells in the respective plates whereas the second group served as a control. The morphological alterations and cell survival were assayed at 24, 48, 72, and 96 hours. Results Morphologically, the initial (10 hours attached fibroblast cells were converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration. The three morphological types of fibroblasts at 48 hours were F-I, F-II and F-III. Aqueous extract of raw arecanut exhibited significant cytotoxicity (p < .0 001 at all time periods studied, when compared against the control values of untreated fibroblasts. Addition of reduced glutathione to cultures showed a significant (p < 0. 001 reduction in cytotoxicity, as indicated by higher optical density values and morphological reversion to the spindle-shaped configuration. CoCONCLUSION:Addition of glutathione reduced the cytotoxic and morphological alterations of the fibroblasts treated with aqueous extracts of both raw and boiled

  3. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  4. Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

    International Nuclear Information System (INIS)

    Rodemann, H.P.; Bayreuther, K.; Francz, P.I.; Dittmann, K.; Albiez, M.

    1989-01-01

    The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern

  5. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

    Science.gov (United States)

    Nayak, Alok Ranjan; Shajahan, T K; Panfilov, A V; Pandit, Rahul

    2013-01-01

    Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac

  6. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    International Nuclear Information System (INIS)

    Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

    2011-01-01

    Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

  7. UV-repair is impaired in fibroblasts from patients with Fanconi's anemia

    International Nuclear Information System (INIS)

    Schwaiger, H.; Hirsch-Kauffmann, M.; Schweiger, M.; Innsbruck Univ.

    1982-01-01

    Fanconi's anemia, a hereditary autosomal disease with chromosomal instability, elevated incidence of cancer and clinical symptoms is accompanied by a DNA repair deficiency. Fibroblasts from patients with Fanconi's anemia were found to be impaired in the DNA repair of UV damage. Nucleoid decondensation and recondensation after UV irradiation were less efficient in fibroblasts from patients with Fanconi's anemia than in those from a healthy proband. These data confirm our earlier findings that DNA ligase is deficient in Fanconi's anemia. (orig.)

  8. The effect of phorbols on metabolic cooperation between human fibroblasts

    International Nuclear Information System (INIS)

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of [ 3 H]hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total [ 3 H]hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of [ 3 H]nucleotides and is an indication of an interference with intercellular communication

  9. Molecular studies of fibroblasts transfected with hepatitis B virus DNA

    International Nuclear Information System (INIS)

    Chen, M.L.; Hood, A.; Thung, S.N.; Gerber, M.A.

    1987-01-01

    Two subclones (D7 and F8) derived from an NIH 3T3 mouse fibroblast cell line after transfection with hepatitis B virus (HBV) genomes, secreted significantly different amounts of HBsAg and HBeAg. DNA extracted from the subclones revealed only integrated and no extrachromosomal HBV DNA sequences as determined by the Southern blot technique with a /sup 32/P-labeled full length HBV DNA probe. The amount and integration sites of HBV sequences were significantly different in the two subclones. HBV DNA sequences coding for HBsAg and HBcAg were detected by alkaline phosphatase-conjugated, single-stranded synthetic gene-specific oligonucleotide probes revealing a larger number of copies in D7 DNA than in F8 DNA. Using a biotinylated probe for in situ hybridization, HBV DNA was found in the nuclei of all D7 cells with predominant localization to a single chromsome, but only in 10-20% of F8 cells. These observations demonstrate different integration patterns of HBV and DNA in two subclones derived from a transfected cell line and suggest that the amount of integrated HBV DNA is proportional to the amount of HBV antigens produced

  10. Human cytomegalovirus replicates in gamma-irradiated fibroblasts

    International Nuclear Information System (INIS)

    Shanley, J.D.

    1986-01-01

    Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication

  11. Fibroblast growth factors as tissue repair and regeneration therapeutics

    Directory of Open Access Journals (Sweden)

    Quentin M. Nunes

    2016-01-01

    Full Text Available Cell communication is central to the integration of cell function required for the development and homeostasis of multicellular animals. Proteins are an important currency of cell communication, acting locally (auto-, juxta-, or paracrine or systemically (endocrine. The fibroblast growth factor (FGF family contributes to the regulation of virtually all aspects of development and organogenesis, and after birth to tissue maintenance, as well as particular aspects of organism physiology. In the West, oncology has been the focus of translation of FGF research, whereas in China and to an extent Japan a major focus has been to use FGFs in repair and regeneration settings. These differences have their roots in research history and aims. The Chinese drive into biotechnology and the delivery of engineered clinical grade FGFs by a major Chinese research group were important enablers in this respect. The Chinese language clinical literature is not widely accessible. To put this into context, we provide the essential molecular and functional background to the FGF communication system covering FGF ligands, the heparan sulfate and Klotho co-receptors and FGF receptor (FGFR tyrosine kinases. We then summarise a selection of clinical reports that demonstrate the efficacy of engineered recombinant FGF ligands in treating a wide range of conditions that require tissue repair/regeneration. Alongside, the functional reasons why application of exogenous FGF ligands does not lead to cancers are described. Together, this highlights that the FGF ligands represent a major opportunity for clinical translation that has been largely overlooked in the West.

  12. Culture conditions affect photoreactivating enzyme levels in human fibroblasts

    International Nuclear Information System (INIS)

    Sutherland, B.M.; Oliver, R.

    1976-01-01

    Photoreactivation of pyrimidine dimers occured under the experimental conditions given in this study, but has not been observed under conditions used by others. Three possible differences were tested in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. The methods in this study of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. It was found that white light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, the cell culture conditions can affect photoreactivating enzyme levels, and thus cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; but companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can reactivate photoreactive cellular dimers

  13. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

    Science.gov (United States)

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. Copyright © 2016. Published by Elsevier B.V.

  14. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

    1979-01-01

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  15. Equibiaxial cyclic stretch stimulates fibroblasts to rapidly remodel fibrin.

    Science.gov (United States)

    Balestrini, Jenna Leigh; Billiar, Kristen Lawrence

    2006-01-01

    Understanding the effects of the mechanical environment on wound healing is critical for developing more effective treatments to reduce scar formation and contracture. The aim of this study was to investigate the effects of dynamic mechanical stretch on cell-mediated early wound remodeling independent of matrix alignment which obscures more subtle remodeling mechanisms. Cyclic equibiaxial stretch (16% stretch at 0.2 Hz) was applied to fibroblast-populated fibrin gel in vitro wound models for eight days. Compaction, density, tensile strength, and collagen content were quantified as functional measures of remodeling. Stretched samples were approximately ten times stronger, eight-fold more dense, and eight times thinner than statically cultured samples. These changes were accompanied by a 15% increase in net collagen but no significant differences in cell number or viability. When collagen crosslinking was inhibited in stretched samples, the extensibility increased and the strength decreased. The apparent weakening was due to a reduction in compaction rather than a decrease in ability of the tissue to withstand tensile forces. Interestingly, inhibiting collagen crosslinking had no measurable effects on the statically cultured samples. These results indicate that amplified cell-mediated compaction and even a slight addition in collagen content play substantial roles in mechanically induced wound strengthening. These findings increase our understanding of how mechanical forces guide the healing response in skin, and the methods employed in this study may also prove valuable tools for investigating stretch-induced remodeling of other planar connective tissues and for creating mechanically robust engineered tissues.

  16. Repair replication in cultured normal and transformed human fibroblasts

    International Nuclear Information System (INIS)

    Smith, C.A.; Hanawalt, P.C.

    1976-01-01

    Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, W138, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labelling method for assaying repair replication. No significant difference was found in the amount of repair replication performed, its dose response, or the time course between growing and confluent W138 cells, early passage and senescent cells, or normal W138 cells and the transformed VA13 cells. When [ 3 H]dThd was employed as the isotopic label in the presence of a 30-200 fold excess of unlabelled BrdUrd apparent differences in repair replication were seen between W138 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [ 3 H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [ 3 H]dThd or a slight discrimination by some cellular process

  17. Comprehensive epigenetic landscape of rheumatoid arthritis fibroblast-like synoviocytes.

    Science.gov (United States)

    Ai, Rizi; Laragione, Teresina; Hammaker, Deepa; Boyle, David L; Wildberg, Andre; Maeshima, Keisuke; Palescandolo, Emanuele; Krishna, Vinod; Pocalyko, David; Whitaker, John W; Bai, Yuchen; Nagpal, Sunil; Bachman, Kurtis E; Ainsworth, Richard I; Wang, Mengchi; Ding, Bo; Gulko, Percio S; Wang, Wei; Firestein, Gary S

    2018-05-15

    Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.

  18. Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells.

    Science.gov (United States)

    Jin, Cheng-Yu; Zhu, Bang-Shang; Wang, Xue-Feng; Lu, Qing-Hua

    2008-09-01

    Nanotitanium dioxide (TiO2) is an important industrial material that is widely used as an additive in cosmetics, pharmaceuticals, and food colorants. Although the small size of the TiO2 nanoparticle is useful in various applications, the biosafety of this material needs to be evaluated. In this study, mouse fibroblast (L929) cells were used to evaluate the cytotoxicity of different concentrations (3-600 microg/mL) of homogeneous and weakly aggregated TiO2 nanoparticles in aqueous solution. The L929 cells became round and even shrank as the concentration of TiO2 nanoparticles increased. Moreover, TiO2 nanoparticle-treated cells had condensed fragmented chromatin or were directly necrosed, as observed by acridine orange (AO) staining. The transmission electron microscopy (TEM) analysis showed that in cells cultured in a medium containing 300 microg/mL TiO2, the number of lysosomes increased, and some cytoplasmic organelles were damaged. In addition, there was a significant increase in oxidative stress at higher TiO2 nanoparticle concentrations (>60 microg/mL). As the concentration of TiO2 nanoparticles increased in the culture medium, the levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) increased, while those of methyl tetrazolium cytotoxicity (MTT), glutathione (GSH), and superoxide dismutase (SOD) decreased. A possible mechanism for the cytotoxicity of TiO2 nanoparticles is also discussed.

  19. MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

    Science.gov (United States)

    Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

    2014-01-01

    Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

  20. Fibroblast Growth Factor 21 Mediates Glycemic Regulation by Hepatic JNK

    Directory of Open Access Journals (Sweden)

    Santiago Vernia

    2016-03-01

    Full Text Available The cJun NH2-terminal kinase (JNK-signaling pathway is implicated in metabolic syndrome, including dysregulated blood glucose concentration and insulin resistance. Fibroblast growth factor 21 (FGF21 is a target of the hepatic JNK-signaling pathway and may contribute to the regulation of glycemia. To test the role of FGF21, we established mice with selective ablation of the Fgf21 gene in hepatocytes. FGF21 deficiency in the liver caused marked loss of FGF21 protein circulating in the blood. Moreover, the protective effects of hepatic JNK deficiency to suppress metabolic syndrome in high-fat diet-fed mice were not observed in mice with hepatocyte-specific FGF21 deficiency, including reduced blood glucose concentration and reduced intolerance to glucose and insulin. Furthermore, we show that JNK contributes to the regulation of hepatic FGF21 expression during fasting/feeding cycles. These data demonstrate that the hepatokine FGF21 is a key mediator of JNK-regulated metabolic syndrome.

  1. Treatment of Skin Avulsion Injuries with Basic Fibroblast Growth Factor

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    Hajime Matsumine, MD, PhD

    2015-04-01

    Full Text Available Summary: This report describes favorable outcomes in 9 patients with skin avulsion injuries of the extremities who underwent full-thickness skin grafting and basic fibroblast growth factor (bFGF application. Following removal of contaminated subcutaneous fat tissue on the inside of skin, the avulsed skin was processed into a full-thickness skin graft, with as much of the skin used as possible irrespective of damage. Several drainage holes (5–10 mm in diameter were made on the graft for drainage from the graft bed and to prevent seroma and hematoma formation. Genetically recombinant human bFGF was sprayed at a dose of 1 μg/cm2 onto the graft bed, which was then covered with the graft and sutured. Pressure immobilization with ointment gauzes and elastic bandages was administered for 1 week postoperatively, and the surface of the skin grafts that did not take was scraped away, preserving the revascularized dermal component on the debrided raw surface as much as possible. bFGF was sprayed again onto the debrided surface to promote epithelialization. Wound closure was achieved in all cases with conservative therapy. The surgical procedure was effective in preventing postoperative ulcer formation and scar contracture and resulted in wound healing with the formation of good-quality, flexible scars.

  2. Variable radiosensitivity in fibroblasts from patients with tuberous sclerosis

    International Nuclear Information System (INIS)

    Hayashi, A.; Yoshida, Y.; Tanaka, H.; Arima, M.; Ohno, K.

    1985-01-01

    It has been reported that some of the cultured cell strains derived from patients with tuberous sclerosis (TS) showed hypersensitivity to gamma-rays or a radiomimetic chemical. Thirteen fibroblast cell strains from 11 patients with TS were examined for their sensitivity to x-rays as determined from their colony-forming ability. All strains derived from normal-appearing skin of patients, either sporadic or familial cases, showed sensitivity within the normal control range. Five cell strains originating from tumorous skin of 3 patients did not show hypersensitivity. It was concluded that the sensitivity to x-rays of cultured cells of TS is essentially normal. However, the mean D0 or D10 values of the strains from tumorous skin tended to be lower compared to those for normal skin of patients. In addition, the hypersensitivity to x-rays was confirmed in the cell strains of TS which had been shown to be hypersensitive to gamma-rays. These results appear to indicate that at least some of the cells of TS are liable to change to exhibit a hypersensitive trait in unknown acquired conditions

  3. DNA repair in a Fanconi's anemia fibroblast cell strain

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

    1979-01-26

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

  4. MicroRNA-mediated myostatin silencing in caprine fetal fibroblasts.

    Directory of Open Access Journals (Sweden)

    Bushuai Zhong

    Full Text Available Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi mediated by microRNAs (miRNAs is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN response genes, such as interferon beta (IFN-β and 2'-5'-oligoadenylate synthetase 2 (OAS2, were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats.

  5. 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

  6. Fibroblast growth factor 10 haploinsufficiency causes chronic obstructive pulmonary disease.

    Science.gov (United States)

    Klar, Joakim; Blomstrand, Peter; Brunmark, Charlott; Badhai, Jitendra; Håkansson, Hanna Falk; Brange, Charlotte Sollie; Bergendal, Birgitta; Dahl, Niklas

    2011-10-01

    Genetic factors influencing lung function may predispose to chronic obstructive pulmonary disease (COPD). The fibroblast growth factor 10 (FGF10) signalling pathway is critical for lung development and lung epithelial renewal. The hypothesis behind this study was that constitutive FGF10 insufficiency may lead to pulmonary disorder. Therefore investigation of the pulmonary functions of patients heterozygous for loss of function mutations in the FGF10 gene was performed. The spirometric measures of lung function from patients and non-carrier siblings were compared and both groups were related to matched reference data for normal human lung function. The patients show a significant decrease in lung function parameters when compared to control values. The average FEV1/IVC quota (FEV1%) for the patients is 0.65 (80% of predicted) and reversibility test using Terbutalin resulted in a 3.7% increase in FEV1. Patients with FGF10 haploinsufficiency have lung function parameters indicating COPD. A modest response to Terbutalin confirms an irreversible obstructive lung disease. These findings support the idea that genetic variants affecting the FGF10 signalling pathway are important determinants of lung function that may ultimately contribute to COPD. Specifically, the results show that FGF10 haploinsufficiency affects lung function measures providing a model for a dosage sensitive effect of FGF10 in the development of COPD.

  7. Functional Diversity of Fibroblast Growth Factors in Bone Formation

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    Yuichiro Takei

    2015-01-01

    Full Text Available The functional significance of fibroblast growth factor (FGF signaling in bone formation has been demonstrated through genetic loss-of-function and gain-of-function approaches. FGFs, comprising 22 family members, are classified into three subfamilies: canonical, hormone-like, and intracellular. The former two subfamilies activate their signaling pathways through FGF receptors (FGFRs. Currently, intracellular FGFs appear to be primarily involved in the nervous system. Canonical FGFs such as FGF2 play significant roles in bone formation, and precise spatiotemporal control of FGFs and FGFRs at the transcriptional and posttranscriptional levels may allow for the functional diversity of FGFs during bone formation. Recently, several research groups, including ours, have shown that FGF23, a member of the hormone-like FGF subfamily, is primarily expressed in osteocytes/osteoblasts. This polypeptide decreases serum phosphate levels by inhibiting renal phosphate reabsorption and vitamin D3 activation, resulting in mineralization defects in the bone. Thus, FGFs are involved in the positive and negative regulation of bone formation. In this review, we focus on the reciprocal roles of FGFs in bone formation in relation to their local versus systemic effects.

  8. Expression of TGF-β3 in Isolated Fibroblasts from Foreskin

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    Mahnaz Mahmoudi Rad

    2015-05-01

    Full Text Available Background: The multifunctional transforming growth factor beta (TGF-β is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3 in human foreskin fibroblasts (HFF’s. Methods: We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA. Results: The highest TGF-β3 expression (8.3-fold greater than control was obtained by lipofection after 72 hours using 3 μl of transfection reagent. Expression was 1.4-fold greater than control by electroporation. Conclusions: In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.

  9. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

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    Elaine Hatanaka

    Full Text Available The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.

  10. Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

    Science.gov (United States)

    Shukla, Vasudha; Barnhouse, Victoria; Ackerman, William E; Summerfield, Taryn L; Powell, Heather M; Leight, Jennifer L; Kniss, Douglas A; Ghadiali, Samir N

    2018-01-01

    The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1β, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1β or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1β induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1β inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

  11. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    Science.gov (United States)

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  12. Decreased Fibroblast and Increased Osteoblast Functions on Ionic Plasma Deposited Nanostructured Ti Coatings

    Directory of Open Access Journals (Sweden)

    Storey Dan

    2007-01-01

    Full Text Available AbstractBioactive coatings are in high demand to control cellular functions for numerous medical devices. The objective of this in vitro study was to characterize for the first time fibroblast (fibrous scar tissue forming cells adhesion and proliferation on an important polymeric biomaterial (silicone coated with titanium using a novel ionic plasma deposition (IPD process. Fibroblasts are one of the first anchorage-dependent cells to arrive at an implant surface during the wound healing process. Persistent excessive functions of fibroblasts have been linked to detrimental fibrous tissue formation which may cause implant failure. The IPD process creates a surface-engineered nanostructure (with features usually below 100 nm by first using a vacuum to remove all contaminants, then guiding charged metallic ions or plasma to the surface of a medical device at ambient temperature. Results demonstrated that compared to currently used titanium and uncoated silicone, silicone coated with titanium using IPD significantly decreased fibroblast adhesion and proliferation. Results also showed competitively increased osteoblast (bone-forming cells over fibroblast adhesion on silicone coated with titanium; in contrast, osteoblast adhesion was not competitively increased over fibroblast adhesion on uncoated silicone or titanium controls. In this manner, this study strongly suggests that IPD should be further studied for biomaterial applications in which fibrous tissue encapsulation is undesirable (such as for orthopedic implants, cardiovascular components, etc..

  13. Emmprin, released as a microvesicle in epithelioid sarcoma, interacts with fibroblasts.

    Science.gov (United States)

    Aoki, Mikiko; Koga, Kaori; Hamasaki, Makoto; Egawa, Nagayasu; Nabeshima, Kazuki

    2017-06-01

    Emmprin (extracellular matrix metalloproteinase inducer, CD147) is a glycosylated transmembrane protein, consisting of two immunoglobulin domains, that stimulates the production of matrix metalloproteinases (MMPs) by tumor-associated fibroblasts. These effects play important roles in tumor invasion and metastasis. However, the precise mechanisms by which emmprin acts on fibroblasts have not been fully elucidated, especially in sarcoma cells. Previously, we demonstrated that emmprin, expressed in conditioned medium collected from the epithelioid sarcoma cell line (FU-EPS-1), stimulates MMP-2 production via interactions with fibroblasts. In this study, we used microvesicles derived from sarcoma cells, and determined whether emmprin exists in the microvesicles, which enhance the production of MMP-2 via fibroblasts. Microvesicles released from FU-EPS-1 cells were shown to contain full-length emmprin, identified as a 45-kDa protein characterized by polylactosamine glycosylation. Microvesicles collected from FU-EPS-1 cells transfected with emmprin-specific siRNA or transduced with shRNA displayed significantly reduced MMP-2 production by fibroblasts compared with those from control-transfected cells. Our findings show that emmprin is released through microvesicle shedding in sarcoma cells, and emmprin in microvesicles regulates MMP-2 production by influencing the activity of fibroblasts located at sites distant from the tumor cells.

  14. Altered Dermal Fibroblasts in Systemic Sclerosis Display Podoplanin and CD90.

    Science.gov (United States)

    Nazari, Banafsheh; Rice, Lisa M; Stifano, Giuseppina; Barron, Alexander M S; Wang, Yu Mei; Korndorf, Tess; Lee, Jungeun; Bhawan, Jag; Lafyatis, Robert; Browning, Jeffrey L

    2016-10-01

    Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34(+) dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34(-), podoplanin(+), and CD90(+) fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1β. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. Blue light-irradiated human keloid fibroblasts: an in vitro study

    Science.gov (United States)

    Magni, Giada; Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Coppi, Elisabetta; Cherchi, Federica; Fusco, Irene; Pugliese, Anna Maria; Pedata, Felicita; Fraccalvieri, Marco; Gasperini, Stefano; Pavone, Francesco S.; Tripodi, Cristina; Alfieri, Domenico; Targetti, Lorenzo

    2018-02-01

    Blue LED light irradiation is currently under investigation because of its effect in wound healing improvement. In this context, several mechanisms of action are likely to occur at the same time, consistently with the presence of different light absorbers within the skin. In our previous studies we observed the wound healing in superficial abrasions in an in vivo murine model. The results evidenced that both inflammatory infiltrate and myofibroblasts activity increase after irradiation. In this study we focused on evaluating the consequences of light absorption in fibroblasts from human cells culture: they play a key role in wound healing, both in physiological conditions and in pathological ones, such as keloid scarring. In particular we used keloids fibroblasts as a new target in order to investigate a possible metabolic or cellular mechanism correlation. Human keloid tissues were excised during standard surgery and immediately underwent primary cell culture extraction. Fibroblasts were allowed to grow in the appropriate conditions and then exposed to blue light. A metabolic colorimetric test (WST-8) was then performed. The tests evidenced an effect in mitochondrial activity, which could be modulated by the duration of the treatment. Electrophysiology pointed out a different behavior of irradiated fibroblasts. In conclusion, the Blue LED light affects the metabolic activity of fibroblasts and thus the cellular proliferation rate. No specific effect was found on keloid fibroblasts, thus indicating a very basic intracellular component, such as cytochromes, being the target of the treatment.

  16. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

    Science.gov (United States)

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-06-07

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.

  17. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    Science.gov (United States)

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

  18. Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro.

    Science.gov (United States)

    Park, Hyun-Jin; Salem, Mabrouka; Semlali, Abdelhabib; Leung, Kai P; Rouabhia, Mahmoud

    2017-07-01

    We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Fibroblast proliferation alters cardiac excitation conduction and contraction: a computational study*

    Science.gov (United States)

    Zhan, He-qing; Xia, Ling; Shou, Guo-fa; Zang, Yun-liang; Liu, Feng; Crozier, Stuart

    2014-01-01

    In this study, the effects of cardiac fibroblast proliferation on cardiac electric excitation conduction and mechanical contraction were investigated using a proposed integrated myocardial-fibroblastic electromechanical model. At the cellular level, models of the human ventricular myocyte and fibroblast were modified to incorporate a model of cardiac mechanical contraction and cooperativity mechanisms. Cellular electromechanical coupling was realized with a calcium buffer. At the tissue level, electrical excitation conduction was coupled to an elastic mechanics model in which the finite difference method (FDM) was used to solve electrical excitation equations, and the finite element method (FEM) was used to solve mechanics equations. The electromechanical properties of the proposed integrated model were investigated in one or two dimensions under normal and ischemic pathological conditions. Fibroblast proliferation slowed wave propagation, induced a conduction block, decreased strains in the fibroblast proliferous tissue, and increased dispersions in depolarization, repolarization, and action potential duration (APD). It also distorted the wave-front, leading to the initiation and maintenance of re-entry, and resulted in a sustained contraction in the proliferous areas. This study demonstrated the important role that fibroblast proliferation plays in modulating cardiac electromechanical behaviour and which should be considered in planning future heart-modeling studies. PMID:24599687

  20. Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

    Directory of Open Access Journals (Sweden)

    Chung-Hsun Chang

    2014-11-01

    Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

  1. Lack of centrioles and primary cilia in STIL(-/-) mouse embryos.

    Science.gov (United States)

    David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

    2014-01-01

    Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL(-/-) mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL(-/-) cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL(-/-) cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

  2. Economy may be harmed by lack of LLW disposal

    International Nuclear Information System (INIS)

    Anon.

    1994-01-01

    A study released by Organizations United for Responsible Low-Level Radioactive Waste Solutions warns that the substantial benefits of using radioactive materials are threatened by the lack of a low-level waste (LLW) disposal facility. The main point of the study is the threat to the American economy posed by insufficient facilities for disposal of the 1.7 billion ft 3 of LLW produced by the use of radioactive materials every year only 34.8 percent of which comes from nuclear power plants. open-quotes Thirty years of experience have provided the technical knowledge to design waste disposal facilities that protect the public and environment. But an impending lack of adequate disposal facilities jeopardizes our continued use of radioactive materials,close quotes according to the study

  3. Lack of diversity in behavioral healthcare leadership reflected in services.

    Science.gov (United States)

    Rosenberg, Linda

    2008-04-01

    America's rapidly changing demographics present an enormous challenge for today's healthcare leaders to redesign the organization and delivery of care to accommodate people who now represent every language, culture and religious belief in the world. So will mental health and addictions services in this country be ready to address the unique needs of these multicultural patients? A survey of the present landscape in 2008 tells us that we have a long, long way to go. Not only are mental health and addictions fields lacking in cultural competency, but there is little diversity in our leadership ranks. Top administrators and executives in behavioral health today are overwhelmingly non-Hispanic whites. This lack of cultural diversity among our leaders will lead to an ever-widening gap in the current chasm of racial and ethnic disparities in healthcare.

  4. Law tightened to protect adults who lack capacity.

    Science.gov (United States)

    2009-05-21

    VULNERABLE OLDER people will be better protected from abuse and poor care after new legislation came into force last month. Under the Mental Capacity Act Deprivation of Liberty Safeguards, a care home or hospital wanting to deprive a person who lacks capacity of their liberty, for their own safety or wellbeing, must now apply for permission. A rigorous, standardised assessment and authorisation process must then be completed.

  5. Return voltage: reproductibility of lack in isolated plastics

    International Nuclear Information System (INIS)

    Frutos, F.; Acedo, M.; Jimenez, A.; Perez, J.A.

    1998-01-01

    Return voltage measures from plane-plane and point-plane experimental test objects of polyethylene are presented. Even though a lack of reproducibility is observed, all the experimental voltage curves can be modellized as the sum of two exponential functions: a first one with a long time period and a second one with a quite shorter time parameter. This analytical behaviour could be theoretically explained by considering an exponential dielectric function response. (Author) 7 refs

  6. The subjetivacion of the lack: between Lacan and Hegel

    Directory of Open Access Journals (Sweden)

    Lorena Souyris Oportot

    2014-05-01

    Full Text Available The present article develops a reflection concerning the figure of the subjectivation and the statute of the lack  in relation to Jacques Lacan y Hegel's thought . The analysis will be addressed from a philosophical approach as and with a psychoanalytic perspective, to show the need to understand the subjectivity, not already as a "work" of duel, but ligature to the loss and the split. The idea is that the above mentioned significances make possible deconstruir and to rethink the duel in lack, that he structures to the subject in an experience "escripturaire" (escriptural and, for the same thing, of dispossession. So that the figure of the subjetivación "in" lack  will allow to grant an important place to the non-place while I spread where the unthinkable thing and the "Autre" registers.  Once exposed this, the reflection will focus on the tragic exigences behind experience “escripturaire” expressed in the image of Antigone

  7. REPROBATION AND LACK OF INTEREST IN MECHATRONICS ENGINEERING STUDENTS

    Directory of Open Access Journals (Sweden)

    César Humberto Guzmán Valdivia

    2013-07-01

    Full Text Available Engineering education in mechatronics is an attractive field of research because it is a new multidisciplinary career. However, a potential problem is the reprobation rate. In the period from January to April 2012 at the Universidad Politécnica de Zacatecas a 53% regular students of a total of 197 were registered. To find the causes of this problem, a survey was conducted to determine the causes of reprobation, lack of motivation and interest to a population of 96 students, of which 40 were the first training cycle, 32 the second and 24 the third. The surveys yielded three main results. The first indicates that the lack of interest is proportional to the time spent in college. The second shows that the reprobation rate is linked to the laziness and the excess of courses. And the last shows a lack of motivation and low expectations of student due to the monotony of the theoretical courses. In conclusion, more research is needed to have a motivated student in an engineering career in mechatronics.

  8. Colony size distributions according to in vitro aging in human skin fibroblasts

    International Nuclear Information System (INIS)

    Kim, Jun Sang; Kim, Jae Sung; Cho, Moon June; Park, Jeong Kyu; Paik, Tae Hyun

    1999-01-01

    To investigate the percentage of colonies with 16 or more cells distribution of human skin fibroblast according to in vitro aging, and to evaluate the relationship between percentage of colonies with 16 or more cells and in vivo donor age in human skin fibroblast culture. C1, C2, C3a, and C3b human skin fibroblast samples from three breast cancer patients were used as subjects. The C1, C2, and C3a donor were 44, 54, and 55 years old, respectively. C3a and C3b cells were isolated from the same person. Single cell suspension of skin fibroblasts was prepared with primary explant technique. One hundred cells are plated into 100ml tissue culture flask and cultured for two weeks. The colony size was defined as colonies with 16 or more cells. The cultured cell was stained with crystal violet, and number of cells in each colony was determined with stereo microscope at x 10 magnification. Passage number of C1, C2, C3a and C3b skin fibroblast were 12th, 17th, and 14th, respectively. Percentage of colonies with 16 or more cells of skin fibroblast samples decreased with increasing in vitro passage number. In contrast, cumulative population doublings of skin fibroblast sample increased with increasing in vitro passage number. Percentage of colonies with 16 or more cells also decreased with increasing population doublings in human skin fibroblast culture. There was strong correlation with percentage of colonised with 16 or more cells and population doublings in C3a skin fibroblast sample. At the same point of population doublings, the percentage of colonies with 16 or more cells of the young C1 donor was higher level than the old C3a donor. The population doublings increased with increasing in vitro passage number but percentage of colonies with 16 or more cells decreased. The results of this study imply that percentage of colonies with 16 or more cells is useful as a indicator of in vitro human skin fibroblast aging and may estimate the in vivo donor age

  9. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  10. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    International Nuclear Information System (INIS)

    Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

    2012-01-01

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

  11. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  12. Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

    International Nuclear Information System (INIS)

    Benamer, Najate; Fares, Nassim; Bois, Patrick; Faivre, Jean-Francois

    2011-01-01

    Highlights: → In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. → S1P increases cell proliferation through SUR2/Kir6.1 activation. → S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. → S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.

  13. Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Benamer, Najate [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Fares, Nassim [Laboratoire de Physiologie, Faculte de Medecine, Universite Saint Joseph, Beyrouth (Lebanon); Bois, Patrick [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Faivre, Jean-Francois, E-mail: Jean-Francois.Faivre@univ-poitiers.fr [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France)

    2011-04-29

    Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

  14. Fibroblast Growth Factor 23 and Kidney Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

    Science.gov (United States)

    Chonchol, Michel; Gitomer, Berenice; Isakova, Tamara; Cai, Xuan; Salusky, Isidro; Pereira, Renata; Abebe, Kaleab; Torres, Vicente; Steinman, Theodor I; Grantham, Jared J; Chapman, Arlene B; Schrier, Robert W; Wolf, Myles

    2017-09-07

    Increases in fibroblast growth factor 23 precede kidney function decline in autosomal dominant polycystic kidney disease; however, the role of fibroblast growth factor 23 in autosomal dominant polycystic kidney disease has not been well characterized. We measured intact fibroblast growth factor 23 levels in baseline serum samples from 1002 participants in the HALT-PKD Study A ( n =540; mean eGFR =91±17 ml/min per 1.73 m 2 ) and B ( n =462; mean eGFR =48±12 ml/min per 1.73 m 2 ). We used linear mixed and Cox proportional hazards models to test associations between fibroblast growth factor 23 and eGFR decline, percentage change in height-adjusted total kidney volume, and composite of time to 50% reduction in eGFR, onset of ESRD, or death. Median (interquartile range) intact fibroblast growth factor 23 was 44 (33-56) pg/ml in HALT-PKD Study A and 69 (50-93) pg/ml in Study B. In adjusted models, annualized eGFR decline was significantly faster in the upper fibroblast growth factor 23 quartile (Study A: quartile 4, -3.62; 95% confidence interval, -4.12 to -3.12 versus quartile 1, -2.51; 95% confidence interval, -2.71 to -2.30 ml/min per 1.73 m 2 ; P for trend kidney volume in adjusted models (quartile 4, 6.76; 95% confidence interval, 5.57 to 7.96 versus quartile 1, 6.04; 95% confidence interval, 5.55 to 6.54; P for trend =0.03). In Study B, compared with the lowest quartile, the highest fibroblast growth factor 23 quartile was associated with elevated risk for the composite outcome (hazard ratio, 3.11; 95% confidence interval, 1.84 to 5.25). Addition of fibroblast growth factor 23 to a model of annualized decline in eGFR≥3.0 ml/min per 1.73 m 2 did not improve risk prediction. Higher serum fibroblast growth factor 23 concentration was associated with kidney function decline, height-adjusted total kidney volume percentage increase, and death in patients with autosomal dominant polycystic kidney disease. However, fibroblast grow