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Sample records for intact fibroblasts endothelial

  1. 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

  2. An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

    Science.gov (United States)

    Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Nyapshaev, I. A.; Kipenko, A. V.; Timoshchuk, K. I.; Podzorova, S. A.; Krylov, B. V.

    2017-02-01

    Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2-10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of 1.7 μm and the average stiffness of the probe-cell contact amounts to 16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

  3. 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...

  4. 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.

  5. In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering

    Directory of Open Access Journals (Sweden)

    T Brune

    2007-12-01

    Full Text Available The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL and cells in an intact ACL (iACL, particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA. Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

  6. Plasma intact fibroblast growth factor 23 levels in women with anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Otani Makoto

    2008-04-01

    Full Text Available Abstract Background Fibroblast growth factor (FGF23 is a novel phosphaturic factor associated with inorganic phosphate homeostasis. Previous human studies have shown that serum FGF23 levels increase in response to a high phosphate diet. For anorexia nervosa (AN patients, inorganic phosphate homeostasis is important in the clinical course, such as in refeeding syndrome. The purpose of this study was to determine plasma levels of intact FGF23 (iFGF23 in restricting-type AN (AN-R patients, binge-eating/purging-type AN (AN-BP patients, and healthy controls. Methods The subjects consisted of 6 female AN-R patients, 6 female AN-BP patients, and 11 healthy female controls; both inpatients and outpatients were included. Plasma iFGF23, 1,25-dihydroxyvitamin D (1,25-(OH2D, and 25-hydroxyvitamin D (25-OHD levels were measured. Data are presented as the median and the range. A two-tailed Mann-Whitney U-test with Bonferroni correction was used to assess differences among the three groups, and a value of p Results There were no differences between AN-R patients and controls in the iFGF23 and 1,25-(OH2D levels. In AN-BP patients, the iFGF23 level (41.3 pg/ml; range, 6.1–155.5 pg/ml was significantly higher than in controls (3.8 pg/ml; range, not detected-21.3 pg/ml; p = 0.001, and the 1,25-(OH2D was significantly lower in AN-BP patients (7.0 pg/ml; range, 4.2–33.7 pg/ml than in controls (39.7 pg/ml; range, 6.3–58.5 pg/ml; p = 0.015. No differences in plasma 25-OHD levels were observed among the groups. Conclusion This preliminary study is the first to show that plasma iFGF23 levels are increased in AN-BP patients, and that these elevated plasma FGF23 levels might be related to the decrease in plasma 1,25-(OH2D levels.

  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.

    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

  8. 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

  9. Regulation by basic fibroblast growth factor of glycosaminoglycan biosynthesis in cultured vascular endothelial cells.

    Science.gov (United States)

    Kaji, T; Hiraga, S; Ohkawara, S; Inada, M; Yamamoto, C; Kozuka, H; Koizumi, F

    1995-05-01

    The alteration of glycosaminoglycans (GAGs) in cultured bovine aortic endothelial cells after exposure to basic fibroblast growth factor (bFGF) was investigated. It was found that the incorporation of [3H]glucosamine into GAGs was markedly increased by bFGF in both the cell layer and the conditioned medium; however, that of [35S]sulfate was not changed by the growth factor. These results indicated that bFGF enhanced the sugar-chain formation but did not affect their sulfation in endothelial GAG production. Similar changes were observed in either bovine aortic smooth-muscle cells and human fibroblastic IMR-90 cells to greater and lesser degrees, respectively. Characterization of GAGs in the endothelial cell layer and the conditioned medium revealed that bFGF enhanced both heparan sulfate and the other GAGs to a similar degree. The present data suggest that bFGF may be involved in the regulation of the blood coagulation system via altering GAGs of the vascular tissue when the endothelium was damaged.

  10. Impairment of lymphocyte adhesion to cultured fibroblasts and endothelial cells by γ-irradiation

    International Nuclear Information System (INIS)

    Piela-Smith, T.H.; Aneiro, L.; Nuveen, E.; Korn, J.H.; Aune, T.

    1992-01-01

    A critical component of immune responsiveness is the localization of effector cells at sites of inflammatory lesions. Adhesive molecules that may play a role in this process have been described on the surfaces of both lymphocytes and connective tissue cells. Adhesive interactions of T lymphocytes with fibroblasts or endothelial cells can be inhibited by preincubation of the fibroblasts or endothelial cells with antibody to intercellular adhesion molecule 1 (CD54) or by preincubation of the T cells with antibody to lymphocyte function-associated Ag 1 (CD11a/CD18), molecules shown to be important in several other cell-cell adhesion interactions. Here the authors show that γ-irradiation of human T lymphocytes impaired their ability to adhere to both fibroblasts and endothelial cells. This impairment was not associated with a loss of cell viability or of cell surface lymphocyte function-associated Ag 1 expression. γ-Irradiation of T cells is known to result in the activation of ADP-ribosyltransferase, an enzyme involved in DNA strand-break repair, causing subsequent depletion of cellular nicotinamide adenine dinucleotide (NAD) pools by increasing NAD consumption for poly(ADP-ribose) formation. Preincubation of T cells with either nicotinamide or 3-aminobenzamide, both known inhibitors of ADP-ribosyltransferase, completely reversed the suppressive effects of γ-irradiation on T cell adhesion. The maintenance of adhesion was accompanied by inhibition of irradiation-induced depletion of cellular NAD. These experiments suggest that the impairment of cellular immune function after irradiation in vivo may be caused, in part, by defective T cell emigration and localization at inflammatory sites. 44 refs., 5 figs., 3 tabs

  11. Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma

    International Nuclear Information System (INIS)

    Jang, F.F.; Wei, W.

    2008-01-01

    Astrocytoma is the most malignant intracranial neoplasm and is characterized by high neovascularization and peritumoural brain oedema. Angiogenesis is a complicated process in oncogenesis regulated by the balance between angiogenic and antiangiogenic factors. The expression of two angiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor were investigated using immunohistochemistry for astrocytoma from 82 patients and 11 normal human tissues. The expression of vascular endothelial growth factor and basic fibroblast growth factor positively correlate with the pathological grade of astrocytoma, microvessel density numbers and brain oedema, which may be responsible for the increased tumour neovascularization and peritumoural brain oedema. The results support the idea that inhibiting vascular endothelial growth factor and basic fibroblast growth factor are useful for the treatment of human astrocytoma and to improve patient's clinical outcomes and prognosis. (author)

  12. Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

    Science.gov (United States)

    Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

    2018-01-01

    Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

  13. Novel GALNT3 mutations causing hyperostosis-hyperphosphatemia syndrome result in low intact fibroblast growth factor 23 concentrations.

    Science.gov (United States)

    Ichikawa, Shoji; Guigonis, Vincent; Imel, Erik A; Courouble, Mélanie; Heissat, Sophie; Henley, John D; Sorenson, Andrea H; Petit, Barbara; Lienhardt, Anne; Econs, Michael J

    2007-05-01

    Hyperostosis-hyperphosphatemia syndrome (HHS) is a rare metabolic disorder characterized by hyperphosphatemia and localized hyperostosis. HHS is caused by mutations in GALNT3, which encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3. Familial tumoral calcinosis (TC), characterized by ectopic calcifications and hyperphosphatemia, is caused by mutations in the GALNT3 or fibroblast growth factor 23 (FGF23) genes. Our objective was to identify mutations in FGF23 or GALNT3 and determine serum FGF23 levels in an HHS patient. Mutation detection in FGF23 and GALNT3 was performed by DNA sequencing, and serum FGF23 concentrations were measured by ELISA. A 5-year-old French boy with HHS and his family members participated. The patient presented with painful cortical lesions in his leg. Radiographs of the affected bone showed diaphyseal hyperostosis. The lesional tissue comprised trabeculae of immature, woven bone surrounded by fibrous tissue. Biochemistry revealed elevated phosphate, tubular maximum rate for phosphate reabsorption per deciliter of glomerular filtrate, and 1,25-dihydroxyvitamin D levels. The patient was a compound heterozygote for two novel GALNT3 mutations. His parents and brother were heterozygous for one of the mutations and had no biochemical abnormalities. Intact FGF23 level in the patient was low normal, whereas C-terminal FGF23 was elevated, a pattern similar to TC. The presence of GALNT3 mutations and elevated C-terminal, but low intact serum FGF23, levels in HHS resemble those seen in TC, suggesting that HHS and TC are different manifestations of the same disorder. The absence of biochemical abnormalities in the heterozygous individuals suggests that one normal allele is sufficient for secretion of intact FGF23.

  14. HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.

    Science.gov (United States)

    Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong

    2013-08-01

    Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

  15. Ablation of the Galnt3 Gene Leads to Low-Circulating Intact Fibroblast Growth Factor 23 (Fgf23) Concentrations and Hyperphosphatemia Despite Increased Fgf23 Expression

    OpenAIRE

    Ichikawa, Shoji; Sorenson, Andrea H.; Austin, Anthony M.; Mackenzie, Donald S.; Fritz, Timothy A.; Moh, Akira; Hui, Siu L.; Econs, Michael J.

    2009-01-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generat...

  16. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    Science.gov (United States)

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

  17. Plasma intact fibroblast growth factor 23 levels in women with bulimia nervosa: A cross-sectional pilot study

    Directory of Open Access Journals (Sweden)

    Yoshiuchi Kazuhiro

    2011-06-01

    Full Text Available Abstract Fibroblast growth factor (FGF 23, a circulating 26-kDa peptide produced by osteogenic cells, is a novel phosphaturic factor. In our previous study, binge-eating/purging type anorexia nervosa (AN-BP patients had elevated plasma intact FGF23 (iFGF23 levels, while restricting type (AN-R patients had plasma iFGF23 levels similar to healthy controls. Although bulimia nervosa (BN patients as well as some patients with AN-BP regularly engage in binge eating, there have been no studies regarding plasma iFGF23 levels in BN patients. Therefore, this study was performed to determine plasma iFGF23 concentrations in BN patients and healthy controls. The study population consisted of 13 female BN patients and 11 healthy female controls. Blood samples were collected from all subjects after overnight fasting. Plasma iFGF23 was measured using an ELISA kit in a cross-sectional manner. The two-tailed Mann-Whitney U-test was used to assess differences between BN patients and healthy controls. In addition, BN patients were divided into two groups based on questionnaire-reported binge eating frequency immediately prior to participation in this study: high frequency of binge eating (once a week or more; HF group; n = 8 and low frequency of binge eating (less than once a week; LF group; n = 5. Two-tailed Mann-Whitney U-test with Bonferroni's correction was performed after the Kruskal-Wallis test to assess differences between HF group, LF group, and healthy controls. Median (quartiles plasma iFGF23 levels were greater in BN patients (35.5 [14.8-65.0] pg/ml than in controls (3.8 [not detected-5.3] pg/ml; p = 0.002. In addition, median (quartiles plasma iFGF23 levels were greater in the HF group (62.3 [44.4-73.4] pg/ml than in controls (p

  18. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.

    Science.gov (United States)

    Ichikawa, Shoji; Sorenson, Andrea H; Austin, Anthony M; Mackenzie, Donald S; Fritz, Timothy A; Moh, Akira; Hui, Siu L; Econs, Michael J

    2009-06-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

  19. 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

  20. Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering

    DEFF Research Database (Denmark)

    Wenger, Andreas; Kowalewski, Nadja; Stahl, Andreas

    2005-01-01

    Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein...... endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid...... to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering...

  1. Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

    Science.gov (United States)

    Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

    1998-04-15

    The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

  2. Ultralarge von Willebrand Factor Fibers Mediate Luminal Staphylococcus aureus Adhesion to an Intact Endothelial Cell Layer Under Shear Stress

    NARCIS (Netherlands)

    Pappelbaum, Karin I.; Gorzelanny, Christian; Graessle, Sandra; Suckau, Jan; Laschke, Matthias W.; Bischoff, Markus; Bauer, Corinne; Schorpp-Kistner, Marina; Weidenmaier, Christopher; Schneppenheim, Reinhard; Obser, Tobias; Sinha, Bhanu; Schneider, Stefan W.

    2013-01-01

    Background During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Methods and Results

  3. Amino acid sequence preferences to control cell-specific organization of endothelial cells, smooth muscle cells, and fibroblasts.

    Science.gov (United States)

    Kanie, Kei; Kato, Ryuji; Zhao, Yingzi; Narita, Yuji; Okochi, Mina; Honda, Hiroyuki

    2011-06-01

    Effective surface modification with biocompatible molecules is known to be effective in reducing the life-threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell-attracting properties on material surfaces, there have been few peptides that could control cell-specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell-specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular-related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell-specific preference was found for amino acids (longer than 5-mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

  4. Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

    Science.gov (United States)

    Shen, S Q; Wang, R; Huang, S G

    2017-03-08

    Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

  5. Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

    Science.gov (United States)

    Zbinden, Aline; Browne, Shane; Altiok, Eda I; Svedlund, Felicia L; Jackson, Wesley M; Healy, Kevin E

    2018-05-01

    Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

  6. Spider angiomas in patients with liver cirrhosis: Role of vascular endothelial growth factor and basic fibroblast growth factor

    Science.gov (United States)

    Li, Chung-Pin; Lee, Fa-Yauh; Hwang, Shinn-Jang; Lu, Rei-Hwa; Lee, Wei-Ping; Chao, Yee; Wang, Sung-Sang; Chang, Full-Young; Whang-Peng, Jacqueline; Lee, Shou-Dong

    2003-01-01

    AIM: To investigate whether vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) are associated with spider angiomas in patients with liver cirrhosis. METHODS: Eighty-six patients with liver cirrhosis were enrolled and the number and size of the spider angiomas were recorded. Fifty-three healthy subjects were selected as controls. Plasma levels of VEGF and bFGF were measured in both the cirrhotics and the controls. RESULTS: Plasma VEGF and bFGF were increased in cirrhotics compared with controls (122 ± 13 vs. 71 ± 11 pg/mL, P = 0.003 for VEGF; 5.1 ± 0.5 vs. 3.4 ± 0.5 pg/mL, P = 0.022 for bFGF). In cirrhotics, plasma VEGF and bFGF were also higher in patients with spider angiomas compared with patients without spider angiomas (185 ± 28 vs. 90 ± 10 pg/mL, P = 0.003 for VEGF; 6.8 ± 1.0 vs. 4.1 ± 0.5 pg/mL, P = 0.017 for bFGF). Multivariate logistic regression showed that young age and increased plasma levels of VEGF and bFGF were the most significant predictors for the presence of spider angiomas in cirrhotic patients (odds ratio [OR] = 6.64, 95% confidence interval [CI] = 2.02-21.79, P = 0.002; OR = 4.35, 95%CI = 1.35-14.01, P = 0.014; OR = 5.66, 95%CI = 1.72-18.63, P = 0.004, respectively). CONCLUSION: Plasma VEGF and bFGF are elevated in patients with liver cirrhosis. Age as well as plasma levels of VEGF and bFGF are significant predictors for spider angiomas in cirrhotic patients. PMID:14669345

  7. Application of chitosan scaffolds on vascular endothelial growth factor and fibroblast growth factor 2 expressions in tissue engineering principles

    Directory of Open Access Journals (Sweden)

    Ariyati Retno Pratiwi

    2015-12-01

    Full Text Available Background: Tissue engineering has given satisfactory results as biological tissue substitutes to restore, replace, or regenerate tissues that have a defect. Chitosan is an organic biomaterial often used in the biomedical field. Chitosan has biocompatible, antifungal, and antibacterial properties. Chitosan is osteoconductive, suitable for bone regeneration applications. Bone defect healing begins with inflammatory phase as a response to the presence of vascular injury, so new vascularization is required. Vascular endothelial growth factor (VEGF and basic fibroblast growth factor-2 (FGF2 are indicators of the beginning of bone regeneration process, playing an important role in angiogenesis. Purpose: This research was aimed to determine the effects of chitosan scaffold application on the expressions of VEGF and FGF2 in tissue engineering principles. Method: Chitosan was dissolved in CH3COOH and NaOH to form a gel. Chitosan gel was then printed in mould to freeze dry for 24 hours. Those rats with defected bones were divided into two groups. Group 1 was the control group which defected bones were not administrated with chitosan scaffolds. Group 2 was the treatment group which defected bones were administrated with chitosan scaffolds. Those rats were sacrificed on day 14. Tissue preparations were made, and then immunohistochemical staining was conducted. Finally, a statistical analysis was conducted using Kruskal Wallis test. Result: There was no significant difference in the expressions of VEGF and FGF2 between the control group and the treatment group (p>0.05. Conclusion: Chitosan scaffolds do not affect the expressions of VEGF and FGF2 during bone regeneration process on day 14 in tissue engineering principles

  8. Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

    Science.gov (United States)

    Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

    2016-10-01

    Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

  9. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

    Science.gov (United States)

    Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

    2003-01-01

    Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

  10. Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

    International Nuclear Information System (INIS)

    Zhang Yueqing; Hu Xiaobo; Tian Ruiyang; Wei Wangui; Hu Wei; Chen Xia; Han Wei; Chen Huayou; Gong Yi

    2006-01-01

    Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the α v -containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism

  11. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    International Nuclear Information System (INIS)

    Laranjeira, Marta S; Carvalho, Ângela; Ferraz, Maria Pia; Monteiro, Fernando Jorge; Pelaez-Vargas, Alejandro; Hansford, Derek; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena

    2014-01-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. (paper)

  12. Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells, fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

    International Nuclear Information System (INIS)

    Iyer, Rohin K; Radisic, Milica; Chiu, Loraine L Y; Vunjak-Novakovic, Gordana

    2012-01-01

    We previously reported that preculture of fibroblasts (FBs) and endothelial cells (ECs) prior to cardiomyocytes (CMs) improved the structural and functional properties of engineered cardiac tissue compared to culture of CMs alone or co-culture of all three cell types. However, these approaches did not result in formation of capillary-like cords, which are precursors to vascularization in vivo. Here we hypothesized that seeding the ECs first on Matrigel and then FBs 24 h later to stabilize the endothelial network (sequential preculture) would enhance cord formation in engineered cardiac organoids. Three sequential preculture groups were tested by seeding ECs (D4T line) at 8%, 15% and 31% of the total cell number on Matrigel-coated microchannels and incubating for 24 h. Cardiac FBs were then seeded (32%, 25% and 9% of the total cell number, respectively) and incubated an additional 24 h. Finally, neonatal rat CMs (60% of the total cell number) were added and the organoids were cultivated for seven days. Within 24 h, the 8% EC group formed elongated cords which eventually developed into beating cylindrical organoids, while the 15% and 31% EC groups proliferated into flat EC monolayers with poor viability. Excitation threshold (ET) in the 8% EC group (3.4 ± 1.2 V cm −1 ) was comparable to that of the CM group (3.3 ± 1.4 V cm −1 ). The ET worsened with increasing EC seeding density (15% EC: 4.4 ± 1.5 V cm −1 ; 31% EC: 4.9 ± 1.5 V cm −1 ). Thus, sequential preculture promoted vascular cord formation and enhanced architecture and function of engineered heart tissues. (paper)

  13. A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

    Science.gov (United States)

    Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

    2009-09-01

    Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

  14. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  15. The impact of hyperbaric oxygen therapy on serological values of vascular endothelial growth factor (VEGF and basic fibroblast growth factor (bFGF

    Directory of Open Access Journals (Sweden)

    Ziebura Thomas

    2010-12-01

    Full Text Available Abstract Background Hyperbaric oxygen (HBO therapy is an effective adjunct treatment for ischemic disorders such as chronic infection or chronic wounds. It combines hyperoxic effects with the stimulating potential of post-therapeutic reactive hypoxia. As its crucial effects, stimulation of fibroblast growth, induction of collagen synthesis and the initiation of angiogenesis are discussed. Angiogenesis is a multistage process resulting in the growth of blood vessels. It includes degradation of extracellular matrix, proliferation and migration of different cell populations and finally formation of new vessel structures. This complex chain of procedures is orchestrated by different cytokines and growth factors. Crucial mediators of angiogenesis are basic fibroblast growth factor (bFGF and vascular endothelial growth factor (VEGF; their in-vivo function is still not fully understood. Methods Forty-three patients suffering from sudden sensorineural hearing loss or tinnitus were treated with HBO. The therapy included 10 sessions of 90 minutes each, one session a day. Serological levels of bFGF and VEGF were assessed by enzyme-linked immunosorbent assays performed according to the manufacturer's instructions on day 1, 2, 5 and 10 of HBO therapy and were compared to mean values of the control group, related to the patient's age and sex, and their development observed over the ten days of HBO. Results There was no sex- or age dependency of bFGF observed in the present study, whereas under HBO our results showed a significant mitigation of the bFGF concentration. In the present data, there was no connection between the VEGF concentration and the patients' ages. Women showed significantly higher levels of VEGF. There was no significant change of VEGF concentration or the VEGF/bFGF ratio during HBO. All scored results varied within the range of standard values as described in the current literature. Conclusions A significant effect of HBO on serum

  16. Fibroblast growth factor-2-induced host stroma reaction during initial tumor growth promotes progression of mouse melanoma via vascular endothelial growth factor A-dependent neovascularization.

    Science.gov (United States)

    Tsunoda, Satoshi; Nakamura, Toshiyuki; Sakurai, Hiroaki; Saiki, Ikuo

    2007-04-01

    Fibroblast growth factor (FGF)-2 has been considered to play a critical role in neovascularization in several tumors; however, its precise role in tumor progression is not fully understood. In the present study, we have characterized the role of FGF-2 in B16-BL6 mouse melanoma cells, focusing on effects during the initial phase of tumor growth. FGF-2 was injected at the tumor inoculation site of dorsal skin during the initial phase. FGF-2 induced marked tumor growth and lymph node metastasis. This was well correlated with an increase in neovascularization in the host stroma. FGF-2 also recruited inflammatory and mesenchymal cells in host stroma. Marked tumor growth, pulmonary metastasis and intensive neovascularization in tumor parenchyma were also observed after a single injection of FGF-2 into the footpad inoculation site. In contrast, repeated injections of FGF-2 at a site remote from the footpad tumor were ineffective in promoting tumor growth and metastasis. These promoting activities of FGF-2 were blocked by local injections of a glucocorticoid hormone, suggesting that host inflammatory responses induced by FGF-2 are associated with FGF-2-induced tumor progression. In addition, although FGF-2 did not promote cellular proliferation and vascular endothelial growth factor A (VEGFA) mRNA expression in B16-BL6 cells in vitro, FGF-2 induced VEGFA expression in host stroma rather than tumor tissue, and local injections of a neutralizing antibody against VEGFA inhibited these activities of FGF-2 in vivo. These results indicate that abundant FGF-2 during the initial phase of tumor growth induces VEGFA-dependent intensive neovascularization in host stroma, and supports marked tumor growth and metastasis.

  17. Atlantic salmon endothelial cells from the heart were more susceptible than fibroblasts from the bulbus arteriosus to four RNA viruses but protected from two viruses by dsRNA pretreatment.

    Science.gov (United States)

    Pham, Phuc H; Tong, Winnie W L; Misk, Ehab; Jones, Ginny; Lumsden, John S; Bols, Niels C

    2017-11-01

    Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

    Directory of Open Access Journals (Sweden)

    Annie O Smith

    Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

  19. Fibroblastic rheumatism

    Directory of Open Access Journals (Sweden)

    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.

  20. Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

    Science.gov (United States)

    Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter

    2004-01-01

    A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

  1. (Photosynthesis in intact plants)

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  2. 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

  3. Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

    Science.gov (United States)

    Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

    2015-01-01

    ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

  4. 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.

  5. Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

    DEFF Research Database (Denmark)

    Wang, X; Häring, M-F; Rathjen, Thomas

    2017-01-01

    in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

  6. Ultrastructure and Light Microscope Analysis of Intact Skin after a Varying Number of Low Level Laser Irradiations in Mice

    Directory of Open Access Journals (Sweden)

    Mamie Mizusaki Iyomasa

    2014-01-01

    Full Text Available Low level laser therapy (LLLT has been used to relieve pain, inflammation, and wound healing processes. Thus, the skin is overexposed to laser and this effect is not completely understood. This study analyzed the effects of the number of laser applications (three, six, and 10 on the intact skin of the masseteric region in mice of strain HRS/J. The animals (n=30 were equally divided into control (0 J/cm2 and irradiated (20 J/cm2, and each of these groups was further equally divided according to the number of laser applications (three, six, and 10 and underwent LLLT on alternate days. Samples were analyzed by light microscopy and transmission electron microscope (TEM. The animals receiving applications exhibited open channels more dilated between the keratinocytes and photobiomodulation effect on endothelial cells and fibroblasts by TEM. Under the light microscope after 10 laser applications, the type I collagen decreased (P<0.05 compared to the three and six applications. Under these experimental conditions, all numbers of applications provided photobiomodulatory effect on the epidermis and dermis, without damage. More studies are needed to standardize the energy density and number of applications recommended for laser therapy to have a better cost-benefit ratio associated with treatment.

  7. Fibroblasts are in a position to provide directional information to migrating neutrophils during pneumonia in rabbit lungs.

    Science.gov (United States)

    Behzad, A R; Chu, F; Walker, D C

    1996-05-01

    Previous findings have shown that pulmonary fibroblasts are associated with preexisting holes in the endothelial and epithelial basal laminae through which neutrophils appear to enter and leave the interstitium as they migrate from capillaries to alveoli. To determine their role in neutrophil migration, fibroblast organization within the interstitium was assessed by transmission electron microscope observations of serial-sectioned rabbit lung tissue. Interstitial fibroblasts were found to physically interconnect the endothelial basal lamina holes to epithelial basal lamina holes. Morphometric assessment of rabbit lung tissue instilled with Streptococcus pneumoniae revealed that approximately 70% of the surface area density of migrating neutrophils is in close contact (15 nm or less) with interstitial fibroblasts and extracellular matrix elements (30 and 40%, respectively). Although migrating neutrophils were close enough to adhere to both fibroblasts and extracellular elements, the interstitial fibroblasts are organized in a manner that would allow them to provide directional information to the neutrophils. A model illustrating this process is proposed.

  8. A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

    Science.gov (United States)

    Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

    2018-03-01

    Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

  9. Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

    2014-01-01

    We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

  10. Radiation-induced inhibition of human endothelial cells replicating in culture

    International Nuclear Information System (INIS)

    DeGowin, R.L.; Lewis, L.J.; Mason, R.E.; Borke, M.K.; Hoak, J.C.

    1976-01-01

    The radiosensitivity of some tumors may depend upon the sensitivity of their microvasculature to radiation. Heretofore, the dose-response of human endothelial cells replicating in tissue culture has not been published. In studies reported here, we exposed flasks containing 4 to 7 x 10 4 genetically identical human endothelial cells to doses of x irradiation from 125 to 1000 rad. During the phase of logarithmic growth, cell counts were compared to those of an unirradiated control to construct a dose--response curve. Similar studies were performed with normal fibroblasts. We found that 160 rad suppressed endothelial cell replication by 37 percent. Although recovery was evident with doses of 500 rad, no net increase in cell number occurred in 3 weeks in flasks of endothelial cells that received 750 or 1000 rad. Fibroblasts were slightly less sensitive under these conditions. To our knowledge, this is the first report of a radiation dose--response curve for human endothelial cells replicating in culture

  11. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    Science.gov (United States)

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  12. 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.

  13. N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

    Directory of Open Access Journals (Sweden)

    Bernadette K. Madathil

    2014-01-01

    Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

  14. 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

  15. Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

    Directory of Open Access Journals (Sweden)

    Cristina Espinosa-Díez

    2018-04-01

    Full Text Available Glutathione (GSH biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL, which is composed of the catalytic (GCLc and the modulatory (GCLm subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice. In murine lung endothelial cells (MLEC derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177 and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+ male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+ mice. We observed that obstructed kidneys from Gclc(e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Keywords: Glutamate-cysteine ligase, ROS, Glutathione, Endothelial dysfunction, Kidney Fibrosis

  16. 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

  17. Strategies for Analyzing Data from Intact Groups.

    Science.gov (United States)

    Cross, Lawrence H.; Lane, Carolyn E.

    Action research often necessitates the use of intact groups for the comparison of educational treatments or programs. This paper considers several analytical methods that might be used for such situations when pretest scores indicate that these intact groups differ significantly initially. The methods considered include gain score analysis of…

  18. Exploratory investigations of hypervelocity intact capture spectroscopy

    Science.gov (United States)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  19. Intact glycopeptide characterization using mass spectrometry

    OpenAIRE

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prykova, Iya; Wu, Si

    2016-01-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to r...

  20. Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

    Science.gov (United States)

    Espinosa-Díez, Cristina; Miguel, Verónica; Vallejo, Susana; Sánchez, Francisco J; Sandoval, Elena; Blanco, Eva; Cannata, Pablo; Peiró, Concepción; Sánchez-Ferrer, Carlos F; Lamas, Santiago

    2018-04-01

    Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH 4 . To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase

    NARCIS (Netherlands)

    Seppen, J.; Tada, K.; Hellwig, S.; Bakker, C. T.; Prasad, V. R.; Roy Chowdhury, N.; Roy Chowdhury, J.; Bosma, P. J.; Oude Elferink, R. P.

    1996-01-01

    Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in

  2. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  3. 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.

  4. Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

    Science.gov (United States)

    Krieg, Thomas; Abraham, David; Lafyatis, Robert

    2007-01-01

    Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

  5. Intact glycopeptide characterization using mass spectrometry.

    Science.gov (United States)

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

    2016-05-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.

  6. 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.

  7. 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

  8. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    International Nuclear Information System (INIS)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-01-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers

  9. 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

  10. 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...

  11. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  12. Key role of the kidney in the regulation of fibroblast growth factor 23

    DEFF Research Database (Denmark)

    Mace, Maria L; Gravesen, Eva; Hofman-Bang, Jacob

    2015-01-01

    was significantly increased in BNX rats. The rapid rise in FGF23 after BNX was independent of parathyroid hormone or FGF receptor signaling. No evidence of early stimulation of FGF23 gene expression in the bone was found. Furthermore, acute severe hyperphosphatemia or hypercalcemia had no impact on intact FGF23......High circulating levels of fibroblast growth factor 23 (FGF23) have been demonstrated in kidney failure, but mechanisms of this are not well understood. Here we examined the impact of the kidney on the early regulation of intact FGF23 in acute uremia as induced by bilateral or unilateral...

  13. Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation.

    Science.gov (United States)

    Ahirwar, Dinesh K; Nasser, Mohd W; Ouseph, Madhu M; Elbaz, Mohamad; Cuitiño, Maria C; Kladney, Raleigh D; Varikuti, Sanjay; Kaul, Kirti; Satoskar, Abhay R; Ramaswamy, Bhuvaneswari; Zhang, Xiaoli; Ostrowski, Michael C; Leone, Gustavo; Ganju, Ramesh K

    2018-05-03

    The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

  14. Transcytosis Involvement in Transport System and Endothelial Permeability of Vascular Leakage during Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Chanettee Chanthick

    2018-02-01

    Full Text Available The major role of endothelial cells is to maintain homeostasis of vascular permeability and to preserve the integrity of vascular vessels to prevent fluid leakage. Properly functioning endothelial cells promote physiological balance and stability for blood circulation and fluid components. A monolayer of endothelial cells has the ability to regulate paracellular and transcellular pathways for transport proteins, solutes, and fluid. In addition to the paracellular pathway, the transcellular pathway is another route of endothelial permeability that mediates vascular permeability under physiologic conditions. The transcellular pathway was found to be associated with an assortment of disease pathogeneses. The clinical manifestation of severe dengue infection in humans is vascular leakage and hemorrhagic diatheses. This review explores and describes the transcellular pathway, which is an alternate route of vascular permeability during dengue infection that corresponds with the pathologic finding of intact tight junction. This pathway may be the route of albumin transport that causes endothelial dysfunction during dengue virus infection.

  15. Interleukin 1 is an autocrine regulator of human endothelial cell growth

    International Nuclear Information System (INIS)

    Cozzolino, F.; Torcia, M.; Aldinucci, D.; Ziche, M.; Bani, D.; Almerigogna, F.; Stern, D.M.

    1990-01-01

    Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, the authors demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G 1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth the suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells

  16. 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.

  17. 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.

  18. Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells.

    Science.gov (United States)

    Davis, K J; Anderson, A O; Geisbert, T W; Steele, K E; Geisbert, J B; Vogel, P; Connolly, B M; Huggins, J W; Jahrling, P B; Jaax, N K

    1997-08-01

    Ebola virus has been responsible for explosive lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Ebola virus for cells of the mononuclear phagocyte system and endothelial cells. To examine the distribution of lesions and Ebola virus antigen in the tissues of six adult male African green monkeys (Cercopithecus aethiops) that died 6 to 7 days after intraperitoneal inoculation of Ebola-Zaire (Mayinga) virus. Tissues were examined histologically, immunohistochemically, and ultrastructurally. A major novel finding of this study was that fibroblastic reticular cells were immunohistochemically and ultrastructurally identified as targets of Ebola virus infection. The role of Ebola virus-infected fibroblastic reticular cells in the pathogenesis of Ebola hemorrhagic fever warrants further investigation. This is especially important because of recent observations indicating that fibroblastic reticular cells, along with the reticular fibers they produce, maximize the efficiency of the immune response.

  19. 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 growth factor 23 did not substantially

  20. Evolution of endothelial keratoplasty.

    Science.gov (United States)

    Price, Francis W; Price, Marianne O

    2013-11-01

    Endothelial keratoplasty has evolved into a popular alternative to penetrating keratoplasty (PK) for the treatment of endothelial dysfunction. Although the earliest iterations were challenging and were not widely adopted, the iteration known as Descemet stripping endothelial keratoplasty (DSEK) has gained widespread acceptance. DSEK combines a simplified technique for stripping dysfunctional endothelium from the host cornea and microkeratome dissection of the donor tissue, a step now commonly completed in advance by eye bank technicians. Studies show that a newer endothelial keratoplasty iteration, known as Descemet membrane endothelial keratoplasty (DMEK), provides an even faster and better visual recovery than DSEK does. In addition, DMEK significantly reduces the risk of immunologic graft rejection episodes compared with that in DSEK or in PK. Although the DMEK donor tissue, consisting of the bare endothelium and Descemet membrane without any stroma, is more challenging to prepare and position in the recipient eye, recent improvements in instrumentation and surgical techniques are increasing the ease and the reliability of the procedure. DSEK successfully mitigates 2 of the main liabilities of PK: ocular surface complications and structural problems (including induced astigmatism and perpetually weak wounds), whereas DMEK further mitigates the 2 principal remaining liabilities of PK: immunologic graft reactions and secondary glaucoma from prolonged topical corticosteroid use.

  1. Trifluoperazine: corneal endothelial phototoxicity

    International Nuclear Information System (INIS)

    Hull, D.S.; Csukas, S.; Green, K.

    1983-01-01

    Trifluoperazine is used for the treatment of psychiatric disorders. Perfusion of corneal endothelial cells with trifluoperazine-HC1 concurrent with exposure to long wavelength ultraviolet light resulted in a corneal swelling rate greater than that found in perfused corneas not exposed to ultraviolet light. Exposure of endothelial cells to 25 W incandescent light during perfusion with trifluoperazine-HC1 did not result in a higher corneal swelling rate compared to those perfused in the dark. The increased corneal swelling rate could be produced by pre-exposure of the trifluoperazine-HC1 perfusing solution to ultraviolet light suggesting the production of toxic photoproducts during exposure of trifluoperazine-HC1 to ultraviolet light. Perfusion of corneal endothelial cells with non-ultraviolet illuminated trifluoperazine-HC1 had no effect on endothelial cell membranes or ultrastructure. This is in contrast to cells perfused with trifluoperazine-HC1 that had been exposed to ultraviolet light in which there was an alteration of mitochondria and a loss of cytoplasmic homogeneity. The data imply that the trifluoperazine-HC1 photoproduct had an adverse effect on cellular transport mechanisms. The study also further demonstrates the value of the corneal endothelial cell model for identifying the physiological and anatomical changes occuring in photo-induced toxic reactions. (author)

  2. Mitochondria and Endothelial Function

    Science.gov (United States)

    Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

    2013-01-01

    In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

  3. 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.

  4. Intacting Integrity in coping with health issues

    DEFF Research Database (Denmark)

    Jepsen, Stine Leegaard; Bastrup Jørgensen, Lene; Fridlund, Bengt

    2017-01-01

    The aim of this study was to develop a formal substantive theory (FST) on the multidimensional behavioral process of coping with health issues. Intacting integrity while coping with health issues emerged as the core category of this FST. People facing health issues strive to safeguard and keep...

  5. Epispadias in boys with an intact prepuce

    NARCIS (Netherlands)

    Bos, E. M. E.; Kuijper, C. F.; Chrzan, R. J.; Dik, P.; Klijn, A. J.; de Jong, T. P. V. M.

    2014-01-01

    To present an overview of the clinical presentation and pathological anatomy, and the results of surgical correction of 7 cases of epispadias with intact prepuce; a rare condition that has only occasionally been reported in literature. A retrospective search was performed in the surgical and

  6. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    Science.gov (United States)

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  7. Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography

    International Nuclear Information System (INIS)

    Stentz, F.B.; Harris, H.L.; Kitabchi, A.E.

    1983-01-01

    We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered ''intact insulin'' by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography

  8. 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

  9. Small Particles Intact Capture Experiment (SPICE)

    Science.gov (United States)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  10. An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.

    Science.gov (United States)

    Rockwell, W B; Ehrlich, H P

    1991-02-01

    Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.

  11. Optical Investigations of Endothelial Cell Motility

    DEFF Research Database (Denmark)

    Rossen, Ninna Struck

    A monolayer of endothelial cells lines the entire circulatory system and create a barrier between the circulatory system and the tissues. To create and maintain an intact barrier, the individual cells have to connect tightly with their neighbors, which causes a highly correlated motion between...... are fascinating from a biophysical point of view. The vasculature also plays a signi cant role in many pathologies. In diabetic blindness or ischemic diseases the ow of blood is insucient to sustain certain tissues or whole limbs. The creation of new blood vessels can relieve or treat such diseases. In other...... pathologies, such as the growth of cancerous tumors and metastasis, the creation of new blood vessels to these tumors worsen the condition and an inhibition of blood vessel creation will relieve the pathology. The thesis is divided into three parts; Part 1 provides some general background knowledge...

  12. 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)

  13. Wine and endothelial function.

    Science.gov (United States)

    Caimi, G; Carollo, C; Lo Presti, R

    2003-01-01

    In recent years many studies have focused on the well-known relationship between wine consumption and cardiovascular risk. Wine exerts its protective effects through various changes in lipoprotein profile, coagulation and fibrinolytic cascades, platelet aggregation, oxidative mechanisms and endothelial function. The last has earned more attention for its implications in atherogenesis. Endothelium regulates vascular tone by a delicate balancing among vasorelaxing (nitric oxide [NO]) and vasoconstrincting (endothelins) factors produced by endothelium in response to various stimuli. In rat models, wine and other grape derivatives exerted an endothelium-dependent vasorelaxing capacity especially associated with the NO-stimulating activity of their polyphenol components. In experimental conditions, reservatrol (a stilbene polyphenol) protected hearts and kidneys from ischemia-reperfusion injury through antioxidant activity and upregulation of NO production. Wine polyphenols are also able to induce the expression of genes involved in the NO pathway within the arterial wall. The effects of wine on endothelial function in humans are not yet clearly understood. A favorable action of red wine or dealcoholized wine extract or purple grape juice on endothelial function has been observed by several authors, but discrimination between ethanol and polyphenol effects is controversial. It is, however likely that regular and prolonged moderate wine drinking positively affects endothelial function. The beneficial effects of wine on cardiovascular health are greater if wine is associated with a healthy diet. The most recent nutritional and epidemiologic studies show that the ideal diet closely resembles the Mediterranean diet.

  14. Infections and endothelial cells

    NARCIS (Netherlands)

    Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

    2003-01-01

    Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

  15. Measurement of diffusive properties of intact rock

    Energy Technology Data Exchange (ETDEWEB)

    Harvey, K B

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada`s Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the `exclusion zone.` A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D{sub o}) for {sup 129}1 and {sup 14}C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D{sup i}) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs.

  16. Measurement of diffusive properties of intact rock

    International Nuclear Information System (INIS)

    Harvey, K.B.

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada's Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the 'exclusion zone.' A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D o ) for 129 1 and 14 C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D i ) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs

  17. Autism Spectrum Disorder and intact executive functioning.

    Science.gov (United States)

    Ferrara, R; Ansermet, F; Massoni, F; Petrone, L; Onofri, E; Ricci, P; Archer, T; Ricci, S

    2016-01-01

    Earliest notions concerning autism (Autism Spectrum Disorders, ASD) describe the disturbance in executive functioning. Despite altered definition, executive functioning, expressed as higher cognitive skills required complex behaviors linked to the prefrontal cortex, are defective in autism. Specific difficulties in children presenting autism or verbal disabilities at executive functioning levels have been identified. Nevertheless, the developmental deficit of executive functioning in autism is highly diversified with huge individual variation and may even be absent. The aim of the present study to examine the current standing of intact executive functioning intact in ASD. Analysis of ASD populations, whether high-functioning, Asperger's or autism Broad Phenotype, studied over a range of executive functions including response inhibition, planning, cognitive flexibility, cognitive inhibition, and alerting networks indicates an absence of damage/impairment compared to the typically-developed normal control subjects. These findings of intact executive functioning in ASD subjects provide a strong foundation on which to construct applications for growth environments and the rehabilitation of autistic subjects.

  18. The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

    Science.gov (United States)

    Mina, Sara G; Huang, Peter; Murray, Bruce T; Mahler, Gretchen J

    2017-07-01

    Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm 2 ) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate

  19. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...

  20. 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.

  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. How can we conserve intact tropical peatlands?

    Science.gov (United States)

    Lawson, Ian; Roucoux, Katherine

    2017-04-01

    The scientific community has, for more than three decades, been expressing increasing alarm about the fate of peatlands in parts of Indonesia and Malaysia, where extensive land-use conversion and drainage for rice and oil palm have greatly compromised peatland hydrology, ecology, biological richness, and carbon storage. The discourse in the literature on these peatlands is now moving on from attempts to preserve the last remaining fragments of peat-swamp forest, towards discussion of how best to restore damaged ecosystems, and whether it is possible to manage plantations more 'sustainably'. It is becoming increasingly clear, however, that peatlands occur quite widely in other parts of the lowland tropics, including parts of Amazonia and the Congo Basin, and many of these peatlands can reasonably be described as 'intact': although few if any parts of the tropics are totally unaffected by human actions, the hydrology and functional ecology of these systems appear to be close to a 'natural' state. The question then arises as to what should be done with the knowledge of their existence. Here we analyse the arguments in favour of protecting intact peatlands, and the potential conflicts with other priorities such as economic development and social justice. We evaluate alternative mechanisms for protecting intact peatlands, focusing on the particular issues raised by peatlands as opposed to other kinds of tropical ecosystem. We identify ways in which natural science agendas can help to inform these arguments, using our own contributions in palaeoecology and carbon mapping as examples. Finally, we argue for a radical reconsideration of research agendas in tropical peatlands, highlighting the potential contribution of methodologies borrowed from the social sciences and humanities.

  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. Sorption of cesium in intact rock

    International Nuclear Information System (INIS)

    Puukko, E.

    2014-04-01

    The mass distribution coefficient K d is used in performance assessment (PA) to describe sorption of a radionuclide on rock. The R d is determined using crushed rock which causes uncertainty in converting the R d values to K d values for intact rock. This work describes a method to determine the equilibrium of sorption on intact rock. The rock types of the planned Olkiluoto waste disposal site were T-series mica gneiss (T-MGN), T-series tonalite granodiorite granite gneiss (T-TGG), P-series tonalite granodiorite granite gneiss (P-TGG) and pegmatitic granite (PGR). These rocks contain different amount of biotite which is the main sorbing mineral. The sorption of cesium on intact rock slices was studied by applying an electrical field to speed up migration of cesium into the rock. Cesium is in the solution as a noncomplex cation Cs + and it is sorbed by ion exchange. The tracer used in the experiments was 134 Cs. The experimental sorption on the intact rock is compared with values calculated using the in house cation exchange sorption model (HYRL model) in PHREEQC program. The observed sorption on T-MGN and T-TGG rocks was close to the calculated values. Two PGR samples were from a depth of 70 m and three samples were from a depth of 150 m. Cesium sorbed more than predicted on the two 70 m PGR samples. The sorption of Cs on the three 150 m PGR samples was small which was consistent with the calculations. The pegmatitic granite PGR has the smallest content of biotite of the four rock types. In the case of P-TGG rock the observed values of sorption were only half of the calculated values. Two kind of slices were cut from P-TGG drill core. The slices were against and to the direction of the foliation of the biotite rims. The sorption of cesium on P-TGG rock was same in both cases. The results indicated that there was no effect of the directions of the electric field and the foliation of biotite in the P-TGG rock. (orig.)

  5. Quantitative proteomic analysis of intact plastids.

    Science.gov (United States)

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  6. Oxidation of molecular tritium by intact soils

    International Nuclear Information System (INIS)

    Sweet, C.W.; Murphy, C.E. Jr.

    1980-01-01

    The effects of environmental factors on the rate of oxidation of molecular tritium (T 2 ) to tritiated water (HTO) were determined for intact soils during field exposures. Maximum deposition velocities of approximately 0.03 cm/sec were measured for T 2 at low wind speeds for a variety of soils over a wide range of conditions. Deposition velocities were slightly inhibited in wet soils and at 0 0 C. In dry soils, oxidation of T 2 to HTO occurred deeper in the soil profile, but deposition velocities were unaffected

  7. 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.

  8. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist; Lorenzen, Janne Kunchel; Gomes, Sisse

    2014-01-01

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC......) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic...

  9. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  10. Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

    Science.gov (United States)

    Cieslik, Katarzyna A; Trial, JoAnn; Crawford, Jeffrey R; Taffet, George E; Entman, Mark L

    2014-05-01

    Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-β), due to reduced expression of TGF-β receptor I (TβRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-β promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-β signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TβRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ". © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  12. Endothelial RIG-I activation impairs endothelial function

    International Nuclear Information System (INIS)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-01-01

    Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  13. Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

    Science.gov (United States)

    Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

    2017-08-01

    Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

  14. CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

    Directory of Open Access Journals (Sweden)

    Gabriele S V Campanella

    2010-09-01

    Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

  15. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

    Science.gov (United States)

    Braundmeier, A G; Dayger, C A; Mehrotra, P; Belton, R J; Nowak, R A

    2012-12-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

  16. 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.

  17. Protein methylation reactions in intact pea chloroplasts

    International Nuclear Information System (INIS)

    Niemi, K.J.

    1989-01-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

  18. A new infusion pathway intactness monitoring system.

    Science.gov (United States)

    Ogawa, Hidekuni; Yonezawa, Yoshiharu; Maki, Hiromichi; Ninomiya, Ishio; Sata, Koji; Hamada, Shingo; Caldwell, W Morton

    2006-01-01

    A new infusion pathway monitoring system has been developed for hospital and home use. The system consists of linear integrated circuits and a low-power 8-bit single chip microcomputer which constantly monitors the infusion pathway intactness. An AC (alternating current) voltage is induced on the patient's body by electrostatic coupling from the normal 100 volt, 60 Hz AC power line wiring field in the patient's room. The induced AC voltage can be recorded by a main electrode wrapped around the infusion polyvinyl chloride tube. A reference electrode is wrapped on the electrode to monitor the AC voltage around the main electrode. If the injection needle or infusion tube becomes detached, then the system detects changes in the induced AC voltages and alerts the nursing station, via the nurse call system or PHS (personal handy phone system).

  19. Reconciling certification and intact forest landscape conservation.

    Science.gov (United States)

    Kleinschroth, Fritz; Garcia, Claude; Ghazoul, Jaboury

    2018-05-29

    In 2014, the Forest Stewardship Council (FSC) added a new criterion to its principles that requires protection of intact forest landscapes (IFLs). An IFL is an extensive area of forest that lacks roads and other signs of human activity as detected through remote sensing. In the Congo basin, our analysis of road networks in formally approved concessionary logging areas revealed greater loss of IFL in certified than in noncertified concessions. In areas of informal (i.e., nonregulated) extraction, road networks are known to be less detectable by remote sensing. Under the current definition of IFL, companies certified under FSC standards are likely to be penalized relative to the noncertified as well as the informal logging sector on account of their planned road networks, despite an otherwise better standard of forest management. This could ultimately undermine certification and its wider adoption, with implications for the future of sustainable forest management.

  20. Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

    Science.gov (United States)

    Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

    2017-09-01

    Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

    Science.gov (United States)

    Anitua, Eduardo; de la Fuente, María; Ferrando, Marcos; Quintana, Fernando; Larreategui, Zaloa; Matorras, Roberto; Orive, Gorka

    2016-11-01

    To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Modeling triple-negative breast cancer heterogeneity: effects of stromal macrophages, fibroblasts and tumor vasculature.

    Science.gov (United States)

    Norton, Kerri-Ann; Jin, Kideok; Popel, Aleksander S

    2018-05-08

    A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates. Copyright © 2018. Published by Elsevier Ltd.

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

    DEFF Research Database (Denmark)

    Maynard, Scott P; Miller, Richard A

    2006-01-01

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

  4. 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.)

  5. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA......Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain...... largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion...

  6. Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: Implications for capillary-like tube formation in a fibrin matrix

    NARCIS (Netherlands)

    Kroon, M.E.; Koolwijk, P.; Vecht, B. van der; Hinsbergh, V.W.M. van

    2000-01-01

    Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth

  7. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

    Science.gov (United States)

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

    2017-09-01

    The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, Pbio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; Pbio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. © The Korean Society for Laboratory Medicine

  8. Fibroblast growth factor signaling potentiates VE-cadherin stability at adherens junctions by regulating SHP2.

    Directory of Open Access Journals (Sweden)

    Kunihiko Hatanaka

    Full Text Available The fibroblast growth factor (FGF system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization.In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity.These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.

  9. Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells.

    Science.gov (United States)

    Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

    2017-01-01

    Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris , Commiphora molmol , Aloe vera , and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgf β1 and Vegf-A genes were monitored by real-time polymerase chain reaction. A. capillus-veneris , C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro . The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts.

  10. Pathophysiology of preterm labor with intact membranes.

    Science.gov (United States)

    Talati, Asha N; Hackney, David N; Mesiano, Sam

    2017-11-01

    Preterm labor with intact membranes is a major cause of spontaneous preterm birth (sPTB). To prevent sPTB a clear understanding is needed of the hormonal interactions that initiate labor. The steroid hormone progesterone acting via its nuclear progesterone receptors (PRs) in uterine cells is essential for the establishment and maintenance of pregnancy and disruption of PR signaling (i.e., functional progesterone/PR withdrawal) is key trigger for labor. The process of parturition is also associated with inflammation within the uterine tissues and it is now generally accepted that inflammatory stimuli from multiple extrinsic and intrinsic sources induce labor. Recent studies suggest inflammatory stimuli induce labor by affecting PR transcriptional activity in uterine cells to cause functional progesterone/PR withdrawal. Advances in understanding the functional interaction of inflammatory load on the pregnancy uterus and progesterone/PR signaling is opening novel areas of research and may lead to rational therapeutic strategies to effectively prevent sPTB. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Excitons in intact cells of photosynthetic bacteria.

    Science.gov (United States)

    Freiberg, Arvi; Pajusalu, Mihkel; Rätsep, Margus

    2013-09-26

    Live cells and regular crystals seem fundamentally incompatible. Still, effects characteristic to ideal crystals, such as coherent sharing of excitation, have been recently used in many studies to explain the behavior of several photosynthetic complexes, especially the inner workings of the light-harvesting apparatus of the oldest known photosynthetic organisms, the purple bacteria. To this date, there has been no concrete evidence that the same effects are instrumental in real living cells, leaving a possibility that this is an artifact of unnatural study conditions, not a real effect relevant to the biological operation of bacteria. Hereby, we demonstrate survival of collective coherent excitations (excitons) in intact cells of photosynthetic purple bacteria. This is done by using excitation anisotropy spectroscopy for tracking the temperature-dependent evolution of exciton bands in light-harvesting systems of increasing structural complexity. The temperature was gradually raised from 4.5 K to ambient temperature, and the complexity of the systems ranged from detergent-isolated complexes to complete bacterial cells. The results provide conclusive evidence that excitons are indeed one of the key elements contributing to the energetic and dynamic properties of photosynthetic organisms.

  12. Positive Youth Development, Life Satisfaction and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    Directory of Open Access Journals (Sweden)

    Daniel Tan Lei Shek

    2013-08-01

    Full Text Available This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies, life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior. Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families.

  13. Endothelial progenitor cell subsets and preeclampsia: Findings and controversies

    Directory of Open Access Journals (Sweden)

    Armin Attar

    2017-10-01

    Full Text Available Vascular remodeling is an essential component of gestation. Endothelial progenitor cells (EPCs play an important role in the regulation of vascular homeostasis. The results of studies measuring the number of EPCs in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of some variations in technical issues and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC, colony forming unit endothelial cells (CFU-ECs, and endothelial colony-forming cells (ECFCs. In general, most studies have shown an increase in the number of CACs in the maternal circulation with a progression in the gestational age in normal pregnancies, while functional capacities measured by CFU-ECs and ECFCs remain intact. In the case of preeclampsia, mobilization of CACs and ECFCs occurs in the peripheral blood of pregnant women, but the functional capacities shown by culture of the derived colony-forming assays (CFU-EC and ECFC assays are altered. Furthermore, the number of all EPC subsets will be reduced in umbilical cord blood in the case of preeclampsia. As EPCs play an important role in the homeostasis of vascular networks, the difference in their frequency and functionality in normal pregnancies and those with preeclampsia can be expected. In this review, there was an attempt to provide a justification for these controversies.

  14. Liposomes and lipid disks traverse the BBB and BBTB as intact forms as revealed by two-step Förster resonance energy transfer imaging

    Directory of Open Access Journals (Sweden)

    Tongcheng Dai

    2018-03-01

    Full Text Available The blood–brain barrier (BBB and the blood–brain tumor barrier (BBTB prevent drug and nano-drug delivery systems from entering the brain. However, ligand-mediated nano-drug delivery systems have significantly enhanced the therapeutic treatment of glioma. In this study we investigated the mechanism especially the integrity of liposomes and lipid disks while traversing the BBB and BBTB both in vitro and in vivo. Fluorophores (DiO, DiI and DiD were loaded into liposomes and lipid disks to form Förster resonance energy transfer (FRET nano-drug delivery systems. Using brain capillary endothelial cells as a BBB model, we show that liposomes and disks are present in the cytoplasm as their intact forms and traverse the BBB with a ratio of 0.68‰ and 1.67‰, respectively. Using human umbilical vein endothelial cells as BBTB model, liposomes and disks remained intact and traversed the BBTB with a ratio of 2.31‰ and 8.32‰ at 3 h. Ex vivo imaging and immunohistochemical results revealed that liposomes and disks could traverse the BBB and BBTB in vivo as intact forms. In conclusion, these observations explain in part the mechanism by which nano-drug delivery systems increase the therapeutic treatment of glioma. KEY WORDS: Liposomes, Disks, Intact form, BBB, BBTB, FRET

  15. MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

    Science.gov (United States)

    Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

    2017-09-01

    Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing.

    Science.gov (United States)

    Xian, Law Jia; Chowdhury, Shiplu Roy; Bin Saim, Aminuddin; Idrus, Ruszymah Bt Hj

    2015-03-01

    Platelet-rich plasma (PRP) has been found to contain a high concentration of growth factors that are present during the process of healing. Studies conducted found that application of PRP accelerates wound healing. In this study, we characterized the skin cell suspension harvested using the co-isolation technique and evaluated the effects of PRP (10% and 20%, v/v) on co-cultured keratinocytes and fibroblasts in terms of wound healing. Human keratinocytes and fibroblasts were harvested via co-isolation technique and separated via differential trypsinization. These cells were then indirectly co-cultured in medium supplemented with 10% or 20% PRP for 3 days without medium change for analysis of wound-healing potential. The wound-healing potential of keratinocytes and fibroblasts was evaluated in terms of growth property, migratory property, extracellular matrix gene expression and soluble factor secretion. The co-isolation technique yielded a skin cell population dominated by fibroblasts and keratinocytes, with a small amount of melanocytes. Comparison between the 10% and 20% PRP cultures showed that the 10% PRP culture exhibited higher keratinocyte apparent specific growth rate, and secretion of hepatocyte growth factor, monocyte chemoattractant protein-1, epithelial-derived neutrophil-activating protein 78 and vascular endothelial growth factor A, whereas the 20% PRP culture has significantly higher collagen type 1 and collagen type 3 expressions and produced more granulocyte-macrophage colony-stimulating factor. PRP concentration modulates keratinocyte and fibroblast wound healing potential, whereby the 10% PRP promotes wound remodeling, whereas the 20% PRP enhances inflammation and collagen deposition. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Papillary endothelial hyperplasia in angiokeratoma.

    Science.gov (United States)

    Mehta, Anurag; Sayal, Satish Kumar; Raman, Deep Kumar; Sood, Aradhana

    2003-01-01

    Papillary endothelial hyperplasia (Masson's tumour) is a reactive proliferation of endothelium producing papillary structures with fibrovascular cores. Dilatation, stasis and accompanying inflammation have been incriminated as the inciting events, evident by the presence of this lesion in haemorrhoids, urethral caruncles and laryngeal polyps. We present here a case of papillary endothelial hyperplasia in angiokeratoma hitherto undescribed despite sharing common etiopathogenetic features of dilatation and stasis with other aforementioned lesions.

  19. Regulation of Ecto-5´-Nucleotidase by Docosahexaenoic Acid in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Vu Thi Thom

    2013-08-01

    Full Text Available Background/Aims: Modulation of extracellular adenine nucleotide and adenosine concentrations is one potential mechanism by which docosahexaenoic acid (DHA may exert beneficial effects in critically ill patients. This study assessed DHA effects on extracellular adenine purines. Methods: Experiments used human pulmonary endothelial cells (HPMEC and umbilical vein endothelial cells (HUVEC treated with DHA (48 h. mRNA level (real-time PCR, expression (western blot, flow cytometry and activities (hydrolysis of etheno(ε-purines and fluorescence HPLC of CD73 (ecto-5´-nucleotidase and CD39 (ecto-NTPDase-1 were quantified. Results: DHA elevated total CD73 membrane protein expression concentration-dependently but CD73 mRNA level did not change. Increased expression was paralleled by increased enzyme activity. Effects observed on membrane level were reversed in intact cells, in which ε-AMP hydrolysis decreased after DHA. In intact endothelial cells ATP release was enhanced and CD39 activity blunted following DHA treatment. Hence, extracellular ATP and ADP concentrations increased and this inhibited ε-AMP hydrolysis. Conclusion: In human endothelial cells DHA caused 1 up-regulation of CD73 protein content and increased AMP hydrolysis at the cell membrane level, 2 increased cellular ATP release, and 3 decreased extracellular ATP/ADP hydrolysis. Thus, reorganization of the extracellular adenine-nucleotide-adenosine axis in response to DHA resulted in an increased extracellular ATP/adenosine ratio.

  20. The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

    Directory of Open Access Journals (Sweden)

    Li Qi-Jing

    2002-06-01

    Full Text Available Abstract Background During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF, is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules. Results Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration. Conclusions These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

  1. Isolation of intact elastin fibers devoid of microfibrils.

    NARCIS (Netherlands)

    Daamen, W.F.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2005-01-01

    Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers

  2. 50 CFR 622.38 - Landing fish intact.

    Science.gov (United States)

    2010-10-01

    ... that is operating under the respective trip limits. Such cut-off fish also may be sold. A maximum of... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish...

  3. Analysis of the receptor-mediated B19V mechanism of internalization in the endothelial B19V infection and adenovirus-mediated reactivation of B19V in endothelial cells

    OpenAIRE

    Pozzuto, Tanja

    2012-01-01

    Human Parvovirus B19 (B19V) is the causative agent of erythema infectiosum, hydrops fetalis, aplastic crises and polyarthritis. B19V displays a very narrow cell and tissue tropism with productive infection thought to be restricted exclusively to erythroid progenitor cells in the bone marrow and fetal liver. However, over the last years increasing evidence for the presence of B19V DNA in other cell types and tissues such as synovial fibroblasts, tonsilles and skin as well as endothelial cells ...

  4. Isolated tumor endothelial cells maintain specific character during long-term culture

    International Nuclear Information System (INIS)

    Matsuda, Kohei; Ohga, Noritaka; Hida, Yasuhiro; Muraki, Chikara; Tsuchiya, Kunihiko; Kurosu, Takuro; Akino, Tomoshige; Shih, Shou-Ching

    2010-01-01

    Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

  5. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    International Nuclear Information System (INIS)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-01-01

    We evaluated the interaction of [ 3 H]1,25(OH) 2 D 3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH) 2 D [1,25(OH) 2 D]. We analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer containing 300 mM KCl and 10 mM sodium molybdate. With normal fibroblasts the affinity and capacity of nuclear uptake of [ 3 H]1,25(OH) 2 D 3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of [ 3 H]1,25(OH) 2 D 3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH) 2 D resulted from five or six distinct genetic mutations in six kindreds

  6. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    International Nuclear Information System (INIS)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-01-01

    The authors evaluated the interaction of [ 3 H]1,25(OH) 2 D3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH) 2 D [1,25(OH) 2 D]. They analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer. With normal fibroblasts the affinity and capacity of nuclear uptake of [ 3 H]1,25(OH) 2 D3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. The following four patterns of interaction with [ 3 H]1,25(OH) 2 D3 were observed with cells cultured from affected patients. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of [ 3 H]1,25(OH) 2 D3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH) 2 D resulted from five or six distinct genetic mutations in six kindreds

  7. Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.

    Science.gov (United States)

    Fetterman, Jessica L; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Duess, Mai-Ann; Farb, Melissa G; Gokce, Noyan; Shirihai, Orian S; Hamburg, Naomi M; Vita, Joseph A

    2016-04-01

    Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Alves, Tercia Rodrigues [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Carvalho da Fonseca, Anna Carolina [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Nunes, Sara Santana; Oliveira da Silva, Aline [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Viana, Nathan Bessa [Universidade Federal do Rio de Janeiro, Laboratorio de Pincas Oticas, Coordenacao de Programas de Estudos Avancados, Instituto de Ciencias Biomedicas, Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro, Instituto de Fisica, Rio de Janeiro (Brazil); Marcondes, Jorge [Universidade Federal do Rio de Janeiro, Hospital Universitario Clementino Fraga Filho, Servico de Neurocirurgia, Rio de Janeiro (Brazil); Legrand, Chantal [Institut Universitaire d' Hematologie, Universite Paris-Diderot, Paris 7, INSERM U553, Paris (France); Moura-Neto, Vivaldo [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); and others

    2011-09-10

    The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

  9. Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

    International Nuclear Information System (INIS)

    Alves, Tercia Rodrigues; Carvalho da Fonseca, Anna Carolina; Nunes, Sara Santana; Oliveira da Silva, Aline; Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad; Viana, Nathan Bessa; Marcondes, Jorge; Legrand, Chantal; Moura-Neto, Vivaldo

    2011-01-01

    The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

  10. 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)

  11. ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2...... toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time-lapse microscopy. We show here that C2 toxin specifically ribosylates - and -actin in both fibroblasts and myofibroblasts. Whereas...

  12. Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

    Directory of Open Access Journals (Sweden)

    Margaret A Schwarz

    Full Text Available Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

  13. Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

    DEFF Research Database (Denmark)

    Brünner, N; Svenstrup, B; Spang-Thomsen, M

    1986-01-01

    An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those...

  14. Endothelial-regenerating cells: an expanding universe.

    Science.gov (United States)

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

    Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

  15. 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.

  16. Sustained apnea induces endothelial activation.

    Science.gov (United States)

    Eichhorn, Lars; Dolscheid-Pommerich, Ramona; Erdfelder, Felix; Ayub, Muhammad Ajmal; Schmitz, Theresa; Werner, Nikos; Jansen, Felix

    2017-09-01

    Apnea diving has gained worldwide popularity, even though the pathophysiological consequences of this challenging sport on the human body are poorly investigated and understood. This study aims to assess the influence of sustained apnea in healthy volunteers on circulating microparticles (MPs) and microRNAs (miRs), which are established biomarkers reflecting vascular function. Short intermittent hypoxia due to voluntary breath-holding affects circulating levels of endothelial cell-derived MPs (EMPs) and endothelial cell-derived miRs. Under dry laboratory conditions, 10 trained apneic divers performed maximal breath-hold. Venous blood samples were taken, once before and at 4 defined points in time after apnea. Samples were analyzed for circulating EMPs and endothelial miRs. Average apnea time was 329 seconds (±103), and SpO 2 at the end of apnea was 79% (±12). Apnea was associated with a time-dependent increase of circulating endothelial cell-derived EMPs and endothelial miRs. Levels of circulating EMPs in the bloodstream reached a peak 4 hours after the apnea period and returned to baseline levels after 24 hours. Circulating miR-126 levels were elevated at all time points after a single voluntary maximal apnea, whereas miR-26 levels were elevated significantly only after 30 minutes and 4 hours. Also miR-21 and miR-92 levels increased, but did not reach the level of significance. Even a single maximal breath-hold induces acute endothelial activation and should be performed with great caution by subjects with preexisting vascular diseases. Voluntary apnea might be used as a model to simulate changes in endothelial function caused by hypoxia in humans. © 2017 Wiley Periodicals, Inc.

  17. Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Ling-Yun Chu

    Full Text Available The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.

  18. STUDIES ON ENDOTHELIAL REACTIONS

    Science.gov (United States)

    Foot, Nathan Chandler

    1923-01-01

    operative. On the other hand, there may be an increase in the phagocytic activity of the endothelium of the sinusoids which might take up more bacteria under these changed conditions. Several investigators have claimed, recently, that there is an increased activity of the liver endothelium following splenectomy, their experiments being directed chiefly toward determining the fate of the erythrocytes. Pearce (1918) in reporting the effects of experimental splenectomy in dogs, states that there are definite compensatory changes in the lymph nodes, in the form of an increased proliferation of endothelial phagocytes, and that the stellate cells of the liver sinusoids often show a similar compensatory increase in number. In both cases the cells are, apparently, formed in situ rather than transported to the organs. He says: ‘Such findings suggest the development of a compensatory function on the part of the lymph-nodes and possibly the liver,’ and suggests that, in times of stress ‘the stellate cells of the liver thus assume, in part at least, the function of destroying red blood-corpuscles by phagocytosis.’ Incidentally, he presents an excellent discussion of the history and subject of splenectomy. Motohashi (1922) reports a great increase in the hemophagic power of the hepatic endothelium and an increase in the number of endothelial elements, after some 45 days following splenectomy in rabbits. Nishikawa and Takagi (1922) have observed similar phenomena with white rats, the Kupffer cells taking up erythrocytes in large numbers in splenectomized animals, whereas controls never show similar propensities on the part of these cells. It may be that different substances cause different reactions on the part of the hepatic endothelium. Contributory Experiment.—A side experiment was performed with five rabbits, two splenectomized and three controls, into which uniform doses of pneumococci were injected intravenously. They all died of septicemia after a few days. The results

  19. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    International Nuclear Information System (INIS)

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-01-01

    Incubation of [ 35 S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35 S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [ 35 S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [ 35 S]heparin proteoglycan after 24 hours and the appearance of free [ 35 S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [ 35 S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

  20. Human diploid fibroblasts have receptors for the globular domain of C1Q

    International Nuclear Information System (INIS)

    Bordin, S.; Page, R.C.

    1986-01-01

    The authors showed that mass cultures of fibroblasts grown from gingival explants in DB medium with 10% human serum are enriched in a phenotype that binds C1q with an affinity much higher than the rest of the population. Because of potential biologic importance of C1q receptors, the authors studied whether the interaction between C1q and this phenotype was mediated by the globular or collagenous domains of the molecule. Globular fragments were prepared by digesting C1q with collagenase, and collagenous fragments obtained after pepsin treatment. C1q binding on cells in suspension was determined by reaction with 125 I-C1q as reported. Competition experiments were performed under conditions in which intact 125 I-C1q binding saturated all available receptors. The results showed that collagenous fragments inhibited 20% of the 125 I-C1q binding to high affinity receptors, whereas inhibition by globular fragments was 70%. Unlabeled intact C1q and collagen type 1 were used as controls, and inhibited 92% and 17% of C1q binding, respectively. These studies show that C1q interacts with the fibroblast phenotype expressing high affinity receptors through its globular domain. The authors suggest that at sites of trauma, native C1 may bind to the surface of these cells via the globular domain of C1q, and that this unique phenotype may play an important role in tissue repair

  1. Zymosterol is located in the plasma membrane of cultured human fibroblasts

    International Nuclear Information System (INIS)

    Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

    1990-01-01

    Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

  2. Vascular Endothelial Growth Factor from Embryonic Status to Cardiovascular Pathology

    Directory of Open Access Journals (Sweden)

    Mohsen Azimi-Nezhad

    2014-05-01

    Full Text Available Vascular endothelial growth factor (VEGF is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF121, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases.

  3. The endothelial border to health

    DEFF Research Database (Denmark)

    Hansen, Nina Wærling; Hansen, Anker Jon; Sams, Anette

    2017-01-01

    player for maintenance of health and for development of a number of diseases. Endothelial dysfunction is known to be an important component of type 2 diabetes, but is also assumed to be involved in many other diseases, for example, rheumatoid arthritis, inflammatory bowel disease, asthma...... extracellular proteins form epitopes for potential specific antibody formation upon interactions with reducing sugars. This paper reviews the endothelial metabolism, biology, inflammatory processes, physical barrier functions, and summarizes evidence that although stochastic in nature, endothelial responses...... to hyperglycemia are major contributors to disease pathophysiology. We present molecular and mechanistic evidence that both biological and physical barriers, protein function, specific immunity, and inflammatory processes are compromised by hyperglycemic events and thus, hyperglycemic events alone should...

  4. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  5. Regional heterogeneity of endothelial cells in the porcine vortex vein system.

    Science.gov (United States)

    Tan, Priscilla Ern Zhi; Yu, Paula K; Cringle, Stephen J; Morgan, William H; Yu, Dao-Yi

    2013-09-01

    The aim of this study was to investigate whether region-dependent endothelial heterogeneity is present within the porcine vortex vein system. The superior temporal vortex vein in young adult pig eyes were dissected out and cannulated. The intact vortex vein system down to the choroidal veins was then perfused with labels for f-actin and nucleic acid. The endothelial cells within the choroidal veins, pre-ampulla, anterior portion of the ampulla, mid-ampulla, posterior portion of the ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein regions were studied in detail using a confocal microscopy technique. The endothelial cell and nuclei length, width, area and perimeter were measured and compared between the different regions. Significant regional differences in the endothelial cell and nuclei length, width, area and perimeter were observed throughout the porcine vortex vein system. Most notably, very narrow and elongated endothelia were found in the post-ampulla region. A lack of smooth muscle cells was noted in the ampulla region compared to other regions. Heterogeneity in endothelial cell morphology is present throughout the porcine vortex vein system and there is a lack of smooth muscle cells in the ampulla region. This likely reflects the highly varied haemodynamic conditions and potential blood flow control mechanisms in different regions of the vortex vein system. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, J.C. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Zheng, G.F. [Department of Vascular Surgery, The People' s Hospital of Ganzhou, Ganzhou (China); Wu, L.; Ou Yang, L.Y.; Li, W.X. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-08-08

    Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs) expressing human basic fibroblast growth factor (hbFGF). After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC), MSCs expressing hbFGF (hbFGF-MSC), MSC controls, and phosphate-buffered saline (PBS) controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF) expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001); however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008) and microvessel density (P<0.001). Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

  7. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

    Directory of Open Access Journals (Sweden)

    J.C. Zhang

    2014-10-01

    Full Text Available Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs expressing human basic fibroblast growth factor (hbFGF. After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC, MSCs expressing hbFGF (hbFGF-MSC, MSC controls, and phosphate-buffered saline (PBS controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001; however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008 and microvessel density (P<0.001. Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

  8. 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.

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  10. Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

    Directory of Open Access Journals (Sweden)

    Jie Zhou

    2016-03-01

    Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

  11. Notoginsenoside Ft1 Promotes Fibroblast Proliferation via PI3K/Akt/mTOR Signaling Pathway and Benefits Wound Healing in Genetically Diabetic Mice.

    Science.gov (United States)

    Zhang, Eryun; Gao, Bo; Yang, Li; Wu, Xiaojun; Wang, Zhengtao

    2016-02-01

    Wound healing requires the essential participation of fibroblasts, which is impaired in diabetic foot ulcers (DFU). Notoginsenoside Ft1 (Ft1), a saponin from Panax notoginseng, can enhance platelet aggregation by activating signaling network mediated through P2Y12 and induce proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells. However, whether it can accelerate fibroblast proliferation and benefit wound healing, especially DFU, has not been elucidated. In the present study on human dermal fibroblast HDF-a, Ft1 increased cell proliferation and collagen production via PI3K/Akt/mTOR signaling pathway. On the excisional wound splinting model established on db/db diabetic mouse, topical application of Ft1 significantly shortened the wound closure time by 5.1 days in contrast with phosphate-buffered saline (PBS) treatment (15.8 versus 20.9 days). Meanwhile, Ft1 increased the rate of re-epithelialization and the amount of granulation tissue at day 7 and day 14. The molecule also enhanced mRNA expressions of COL1A1, COL3A1, transforming growth factor (TGF)-β1 and TGF-β3 and fibronectin, the genes that contributed to collagen expression, fibroblast proliferation, and consequent scar formation. Moreover, Ft1 facilitated the neovascularization accompanied with elevated vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor at either mRNA or protein levels and alleviated the inflammation of infiltrated monocytes indicated by reduced tumor necrosis factor-α and interleukin-6 mRNA expressions in the diabetic wounds. Altogether, these results indicated that Ft1 might accelerate diabetic wound healing by orchestrating multiple processes, including promoting fibroblast proliferation, enhancing angiogenesis, and attenuating inflammatory response, which provided a great potential application of it in clinics for patients with DFU. Copyright © 2016 by The American Society for Pharmacology and

  12. 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

  13. 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.

  14. 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.

  15. Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

    Science.gov (United States)

    Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

    2009-02-01

    Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

  16. Endothelial dysfunction after non-cardiac surgery

    DEFF Research Database (Denmark)

    Søndergaard, E S; Fonnes, S; Gögenur, I

    2015-01-01

    was to systematically review the literature to evaluate the association between non-cardiac surgery and non-invasive markers of endothelial function. METHODS: A systematic search was conducted in MEDLINE, EMBASE and Cochrane Library Database according to the PRISMA guidelines. Endothelial dysfunction was described only...... transplantation and vascular surgery respectively) had an improvement in endothelial dysfunction 1 month after surgery. CONCLUSION: Endothelial function changes in relation to surgery. Assessment of endothelial function by non-invasive measures has the potential to guide clinicians in the prevention or treatment...

  17. 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...

  18. 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

  19. Essential Role of Endothelial Notch1 in Angiogenesis

    Science.gov (United States)

    Limbourg, Florian P.; Takeshita, Kyosuke; Radtke, Freddy; Bronson, Roderick T.; Chin, Michael T.; Liao, James K.

    2009-01-01

    Background Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling. Methods and Results We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1flox/flox mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube. Conclusions These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis. PMID:15809373

  20. Endothelial dysfunction: a comprehensive appraisal

    Directory of Open Access Journals (Sweden)

    Vilariño Jorge O

    2006-02-01

    Full Text Available Abstract The endothelium is a thin monocelular layer that covers all the inner surface of the blood vessels, separating the circulating blood from the tissues. It is not an inactive organ, quite the opposite. It works as a receptor-efector organ and responds to each physical or chemical stimulus with the release of the correct substance with which it may maintain vasomotor balance and vascular-tissue homeostasis. It has the property of producing, independently, both agonistic and antagonistic substances that help to keep homeostasis and its function is not only autocrine, but also paracrine and endocrine. In this way it modulates the vascular smooth muscle cells producing relaxation or contraction, and therefore vasodilatation or vasoconstriction. The endothelium regulating homeostasis by controlling the production of prothrombotic and antithrombotic components, and fibrynolitics and antifibrynolitics. Also intervenes in cell proliferation and migration, in leukocyte adhesion and activation and in immunological and inflammatory processes. Cardiovascular risk factors cause oxidative stress that alters the endothelial cells capacity and leads to the so called endothelial "dysfunction" reducing its capacity to maintain homeostasis and leads to the development of pathological inflammatory processes and vascular disease. There are different techniques to evaluate the endothelium functional capacity, that depend on the amount of NO produced and the vasodilatation effect. The percentage of vasodilatation with respect to the basal value represents the endothelial functional capacity. Taking into account that shear stress is one of the most important stimulants for the synthesis and release of NO, the non-invasive technique most often used is the transient flow-modulate "endothelium-dependent" post-ischemic vasodilatation, performed on conductance arteries such as the brachial, radial or femoral arteries. This vasodilatation is compared with the

  1. 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...

  2. 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.

  3. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    International Nuclear Information System (INIS)

    Amann, F.; Vogelhuber, M.

    2015-11-01

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature

  4. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    Energy Technology Data Exchange (ETDEWEB)

    Amann, F. [Eidgenössische Technische Hochschule ETHZ, Zürich (Switzerland); Vogelhuber, M. [Dr. von Moos AG, Geotechnisches Büro, Zürich (Switzerland)

    2015-11-15

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature.

  5. Challenges in pediatric endothelial keratoplasty

    Directory of Open Access Journals (Sweden)

    Vikas Mittal

    2014-01-01

    Full Text Available We performed endothelial keratoplasty (EK in three eyes of two siblings (2.5 years, male and 3.5 years, female with congenital hereditary endothelial dystrophy (CHED and report the intraoperative and postoperative difficulties. Repeated iris prolapse, apprehension of crystalline lens touch due to positive vitreous pressure, and need for frequent air injections to attach the graft were intraoperative challenges in all three eyes. These were addressed by use of Sheet′s glide instead of Busin′s glide during graft insertion and suturing of main and side ports before air injection. One eye had graft dislocation on second postoperative day due to eye rubbing by the child. Graft was repositioned with air and a venting incision was created. Postoperative examination required repeated general anesthesia. Corneal edema resolved completely in all three eyes. Present case series highlights the possible intraoperative and postoperative challenges and their solutions in pediatric EK for CHED.

  6. Lung damage and pulmonary uptake of serotonin in intact dogs

    International Nuclear Information System (INIS)

    Dawson, C.A.; Christensen, C.W.; Rickaby, D.A.; Linehan, J.H.; Johnston, M.R.

    1985-01-01

    The authors examined the influence of glass bead embolization and oleic acid, dextran, and imipramine infusion on the pulmonary uptake of trace doses of [ 3 H]serotonin and the extravascular volume accessible to [ 14 C]antipyrine in anesthetized dogs. Embolization and imipramine decreased serotonin uptake by 53 and 61%, respectively, but no change was observed with oleic acid or dextran infusion. The extravascular volume accessible to the antipyrine was reduced by 77% after embolization and increased by 177 and approximately 44% after oleic acid and dextran infusion, respectively. The results suggest that when the perfused endothelial surface is sufficiently reduced, as with embolization, the uptake of trace doses of serotonin will be depressed. In addition, decreases in serotonin uptake in response to imipramine in this study and in response to certain endothelial toxins in other studies suggest that serotonin uptake can reveal certain kinds of changes in endothelial function. However, the lack of a response to oleic acid-induced damage in the present study suggests that serotonin uptake is not sensitive to all forms of endothelial damage

  7. Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17.

    Directory of Open Access Journals (Sweden)

    Neil Hoa

    Full Text Available Insulin-like growth factor-1 receptor (IGF-1R comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD. When activated by IGF-1 or GD-derived IgG (GD-IgG, these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125I IGF-1, (125I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.

  8. Polyphenols in preventing endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Sylwia Biegańska-Hensoldt

    2017-03-01

    Full Text Available One of the main causes of mortality in developed countries is atherosclerosis. The pathogenesis of atherosclerosis is associated with endothelial dysfunction. Consumption of food rich in natural antioxidants including polyphenols significantly improves endothelial cells functions.Polyphenols have a beneficial effect on the human body and play an important part in protecting the cardiovascular system. Polyphenols present in food have antioxidant, anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. Catechins cause an increase in the activity of endothelial nitric oxide synthase (eNOS and increased production of nitric oxide (NO and decrease in blood pressure. Catechins also reduce platelet adhesion, lower the concentration of C-reactive protein and tumor necrosis factor alpha and interleukin-6. Resveratrol inhibits NADPH oxidase expression, increases the expression of eNOS and NO production as well as decreases the expression of proinflammatory cytokines, and also lowers the concentration of the soluble forms of adhesion molecules – sICAM-1 and sVCAM-1 in blood. Quercetin reduces the blood level of low density lipoprotein cholesterol, lowers blood pressure, reduces the concentration of C-reactive protein and F2-isoprostane level. Curcumin has antagonistic activity to homocysteine. Curcumin increases the expression of eNOS and reduces oxidative DNA damage in rat cardiomyocytes. Numerous attempts are taken for improving the bioavailability of polyphenols in order to increase their use in the body.

  9. Fibroblast Growth Factor-23—A Potential Uremic Toxin

    Directory of Open Access Journals (Sweden)

    Piotr Kuczera

    2016-12-01

    Full Text Available Fibroblast growth factor-23 (FGF23 is a circulating member of the FGF family produced mainly by the osteocytes and osteoblasts that can act as a hormone. The main action of FGF23 is to lower phosphatemia via the reduction of urinary phosphate reabsorption and the decrease of 1,25(OH2-D generation in the kidney. In the course of chronic kidney disease (CKD, plasma FGF23 concentration rises early, most probably to compensate the inability of the deteriorating kidneys to excrete an adequate amount of phosphate. However, this comes at the cost of FGF23-related target organ toxicity. Results of clinical studies suggest that elevated plasma FGF23 concentration is independently associated with the increased risk of CKD progression, occurrence of cardio-vascular complications, and mortality in different stages of CKD. FGF23 also contributes to cardiomyocyte hypertrophy, vascular calcification, and endothelial dysfunction. The impact of FGF23 on heart muscle is not dependent on Klotho, but rather on the PLCγ–calcineurin–NFAT (nuclear factor of activated T-cells pathway. Among the factors increasing plasma FGF23 concentration, active vitamin D analogues play a significant role. Additionally, inflammation and iron deficiency can contribute to the increase of plasma FGF23. Among the factors decreasing plasma FGF23, dietary phosphate restriction, some intestinal phosphate binders, cinacalcet (and other calcimimetics, and nicotinamide can be enumerated. Anti-FGF23 antibodies have also recently been developed to inhibit the action of FGF23 in target organs. Still, the best way to normalize plasma FGF23 in maintenance hemodialysis patients is restoring kidney function by successful kidney transplantation.

  10. Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

    International Nuclear Information System (INIS)

    Majkova, Zuzana; Smart, Eric; Toborek, Michal; Hennig, Bernhard

    2009-01-01

    Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R -/- mice (control) or caveolin-1 -/- /LDL-R -/- mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1 -/- /LDL-R -/- mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor α-naphthoflavone (α-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.

  11. The Role of Plasma Transfusion in Massive Bleeding: Protecting the Endothelial Glycocalyx?

    Directory of Open Access Journals (Sweden)

    Stefano Barelli

    2018-04-01

    Full Text Available Massive hemorrhage is a leading cause of death worldwide. During the last decade several retrospective and some prospective clinical studies have suggested a beneficial effect of early plasma-based resuscitation on survival in trauma patients. The underlying mechanisms are unknown but appear to involve the ability of plasma to preserve the endothelial glycocalyx. In this mini-review, we summarize current knowledge on glycocalyx structure and function, and present data describing the impact of hemorrhagic shock and resuscitation fluids on glycocalyx. Animal studies show that hemorrhagic shock leads to glycocalyx shedding, endothelial inflammatory changes, and vascular hyper-permeability. In these animal models, plasma administration preserves glycocalyx integrity and functions better than resuscitation with crystalloids or colloids. In addition, we briefly present data on the possible plasma components responsible for these effects. The endothelial glycocalyx is increasingly recognized as a critical component for the physiological vasculo-endothelial function, which is destroyed in hemorrhagic shock. Interventions for preserving an intact glycocalyx shall improve survival of trauma patients.

  12. Krüppel-Like Factor 4 Is a Regulator of Proinflammatory Signaling in Fibroblast-Like Synoviocytes through Increased IL-6 Expression

    Directory of Open Access Journals (Sweden)

    Xinjing Luo

    2016-01-01

    Full Text Available Human fibroblast-like synoviocytes play a vital role in joint synovial inflammation in rheumatoid arthritis (RA. Proinflammatory cytokines induce fibroblast-like synoviocyte activation and dysfunction. The inflammatory mediator Krüppel-like factor 4 is upregulated during inflammation and plays an important role in endothelial and macrophage activation during inflammation. However, the role of Krüppel-like factor 4 in fibroblast-like synoviocyte activation and RA inflammation remains to be defined. In this study, we identify the notion that Krüppel-like factor 4 is higher expressed in synovial tissues and fibroblast-like synoviocytes from RA patients than those from osteoarthritis patients. In vitro, the expression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes is induced by proinflammatory cytokine tumor necrosis factor-α. Overexpression of Krüppel-like factor 4 in RA fibroblast-like synoviocytes robustly induced interleukin-6 production in the presence or absence of tumor necrosis factor-α. Conversely, knockdown of Krüppel-like factor 4 markedly attenuated interleukin-6 production in the presence or absence of tumor necrosis factor-α. Krüppel-like factor 4 not only can bind to and activate the interleukin-6 promoter, but also may interact directly with nuclear factor-kappa B. These results suggest that Krüppel-like factor 4 may act as a transcription factor mediating the activation of fibroblast-like synoviocytes in RA by inducing interleukin-6 expression in response to tumor necrosis factor-α.

  13. Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

    Science.gov (United States)

    Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

    2017-01-01

    Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

  14. 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.

  15. Bioavailability and in vivo metabolism of intact glucosinolates

    DEFF Research Database (Denmark)

    Sørensen, Jens Christian; Frandsen, Heidi Blok; Jensen, Søren Krogh

    2016-01-01

    Health benefits associated with consumption of cruciferous vegetables have received considerable attention with a hitherto focus on the role and bioactivity of glucosinolate degradation products. We investigated the in vivo metabolism of intact glucosinolates by following their fate in digesta an...

  16. Capillary zone electrophoresis-mass spectromet of intact proteins

    NARCIS (Netherlands)

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W.

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS

  17. Intact collagen and atelocollagen sponges: Characterization and ESEM observation

    International Nuclear Information System (INIS)

    Ruozi, Barbara; Tosi, Giovanni; Leo, Eliana; Parma, Bruna; Vismara, Susanna; Forni, Flavio; Vandelli, Maria Angela

    2007-01-01

    In this study we have investigated the chemical-physical and morphological properties of intact and atelocollagen sponges used for tissue engineering. The porous sponges were prepared by lyophilization and their physico-chemical characteristics (water binding capacity, denaturing temperature, amino group content) were investigated. Considering the importance of the 'in vivo' interactions between these sponges and the tissue, our attention was addressed (a) to clarify the relationships between the morphology and the amount of water absorbed and (b) to evaluate the influence of pepsin-alkaline treatment on the reorganization of the atelocollagen fibres. Conventional scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) were employed to study the morphology and wetting behaviour of the intact and atelocollagen sponges. The observations by SEM indicated remarkable differences both in the structure and dimension of the pores between intact and atelocollagen sponges. At the data are related to a different water binding capacity. However, the ESEM observations, achieved by changing the relative humidity in the operative chamber, demonstrated that the water adsorbed can be removed with major difficulty from atelocollagen sponges than from intact ones

  18. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    Johnson, R.M.; Nigro, M.

    1986-01-01

    The uptake of exogenous 32 Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[ 32 P]ATP, and suggests a possible reinterpretation of those experiments

  19. Behavior Management Style of Single Parents and Intact Families.

    Science.gov (United States)

    Smith, Douglas K.; And Others

    Studies examining the behavior management styles of parents as a function of family intactness and parent employment status are lacking. To assess parental style of behavior management, the Parental Management Questionnaire (PMQ) was completed by 1,957 parents of elementary school children (50% response rate). The PMQ is based on Aronfreed's…

  20. Non-intact zona improves development of murine preimplantation ...

    African Journals Online (AJOL)

    ajl5

    2012-09-25

    Sep 25, 2012 ... 2College of Animal Science and Technology, Northwest A & F University, Yangling, ... Key words: Mouse, non-intact zona embryos, adenovirus vector with green fluorescent protein (pAd-GFP), .... Based on microscopic examination, the ZP of some ..... permeable structure of ZP that allowed penetration of.

  1. Pharmacokinetics of erythropoietin in intact and anephric dogs

    International Nuclear Information System (INIS)

    Fu, J.S.; Lertora, J.J.; Brookins, J.; Rice, J.C.; Fisher, J.W.

    1988-01-01

    The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp was significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs

  2. Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

    Science.gov (United States)

    Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

    2013-05-03

    Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial

  3. Threats to intact tropical peatlands and opportunities for their conservation.

    Science.gov (United States)

    Roucoux, K H; Lawson, I T; Baker, T R; Del Castillo Torres, D; Draper, F C; Lähteenoja, O; Gilmore, M P; Honorio Coronado, E N; Kelly, T J; Mitchard, E T A; Vriesendorp, C F

    2017-12-01

    Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza-Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land-use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon-dense (domed pole forest) areas. New carbon-based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development. © 2017 The Authors. Conservation Biology published by Wiley Periodicals, Inc

  4. Resveratrol: A Multifunctional Compound Improving Endothelial Function

    OpenAIRE

    Li, Huige; F?rstermann, Ulrich

    2009-01-01

    The red wine polyphenol resveratrol boosts endothelium-dependent and -independent vasorelaxations. The improvement of endothelial function by resveratrol is largely attributable to nitric oxide (NO) derived from endothelial NO synthase (eNOS). By stimulating eNOS expression, eNOS phosphorylation and eNOS deacetylation, resveratrol enhances endothelial NO production. By upregulating antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and suppressing the expression a...

  5. Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

    Science.gov (United States)

    Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

    2015-01-01

    As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the

  6. α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

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

    Science.gov (United States)

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

    2015-01-01

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

  8. Signaling hierarchy regulating human endothelial cell development.

    Science.gov (United States)

    Kelly, Melissa A; Hirschi, Karen K

    2009-05-01

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

  9. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

    International Nuclear Information System (INIS)

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua; Cheng Guoxiang

    2005-01-01

    Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo r ), replaced the α-lactalbumin gene in a 210 kb human α-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock

  10. TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    International Nuclear Information System (INIS)

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-01-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway

  11. Expression of Vascular Endothelial Growth Factor in Odontogenic Cysts: Is There Any Impression on Clinical Outcome?

    Science.gov (United States)

    Sadri, Donia; Farhadi, Sareh; Shahabi, Zahra; Sarshar, Samaneh

    2016-01-01

    The recent scientific reports have shown that angiogenesis can affect biological behavior of pathologic lesions. Regarding unique clinical outcome of Odontogenic keratocyst (OKC), the present study was aimed to compare angiogenesis in Odontogenic keratocyst and Dentigerous cyst (DC). In this experimental study, tissue sections of 46 samples of OKC and DC were stained through immunohistochemical method using Vascular Endothelial Growth Factor (VEGF) antibody. VEGF expression was evaluated in epithelial cells, fibroblasts and endothelial cells. The average percentage of stained cells in any samples was categorized to 3 groups as follows: SCORE 0: 10% of cells or less are positive. SCORE 1: 10 to 50% of cells are positive. SCORE 2: more than 50% of cells are positive. Mann-U-Whitney, T-test and chi-square was used for statistical analysis. The average of VEGF expression in 24 samples of DC was 20.2% and in 22 samples of OKC was 52.6%, respectively. The average of VEGF expression in these two cysts had statistical significant differences. (PV= 0.045). There was significant statistical differences between two cysts in the terms of VEGF SCORE (PV= 0.000). OKC samples had significantly higher SCORE for the purpose of VEGF incidence than DC. Also, there were no differences between VEGF expression in epithelial cells of two cysts (PV= 0.268) there were significant statistical differences between two cysts in terms of endothelial cell staining. The endothelial cell staining was significantly higher in OKC than DC (PV= 0.037%). Regarding higher expression of Vascular Endothelial Growth factor in OKC than DC, it seems that angiogenesis may have great impression on clinical outcome of OKC.

  12. Synergistic inhibition of endothelial cell proliferation, tube formation, and sprouting by cyclosporin A and itraconazole.

    Directory of Open Access Journals (Sweden)

    Benjamin A Nacev

    Full Text Available Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA, an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.

  13. Synergistic inhibition of endothelial cell proliferation, tube formation, and sprouting by cyclosporin A and itraconazole.

    Science.gov (United States)

    Nacev, Benjamin A; Liu, Jun O

    2011-01-01

    Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50) dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.

  14. 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...

  15. 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.

  16. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    Science.gov (United States)

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  17. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe and Explain Differences Between Intact Groups

    NARCIS (Netherlands)

    Schoonenboom, J.I.

    2014-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a

  18. Biodegradable poly (lactic acid-co-glycolic acid scaffolds as carriers for genetically-modified fibroblasts.

    Directory of Open Access Journals (Sweden)

    Tatjana Perisic

    Full Text Available Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid (PLGA scaffolds (Vicryl & Ethisorb as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP and vascular endothelial growth factor (VEGF165 and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.

  19. 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.

  20. Pathways of sphingomyelin metabolism in cultured fibroblasts from normal and sphingomyelin lipidosis subjects.

    Science.gov (United States)

    Spence, M W; Clarke, J T; Cook, H W

    1983-07-25

    The metabolism of endogenous sphingomyelin labeled with 32P or [methyl-3H]choline and of exogenous [choline-methyl-3H], [32P]-, or [N-acyl-1-14C]sphingomyelin was studied in normal and Niemann-Pick Type A (NP-A) cultured fibroblasts. Despite a greater than 96% decrease in lysosomal sphingomyelinase activity in the NP-A cells, they were able to degrade endogenously produced [32P]- or [methyl-3H]sphingomyelin at normal or near normal rates. Exogenous [methyl-3H]-, [methyl-3H, 32P]-, and [methyl-3H, N-acyl-1-14C] sphingomyelin was taken up intact by normal and NP-A cells, with NP-A cells accumulating 4-8 times more lipid. By 20 h, 50% of the control cell-associated 3H and 32P was recovered in lecithin, and the ratio of activities (3H/32P) indicated most of the phosphorylcholine derived from sphingomyelin had been transferred intact. By comparison in NP-A cells, after a 40-h incubation only 20% of the labeled phosphorylcholine derived from sphingomyelin was recovered in lecithin. With both cell lines, 20 to 50 times more sphingomyelin was hydrolyzed than was taken up by the cells; the reaction products in the medium were ceramide and a mixture of water-soluble compounds such as phosphorylcholine and choline. These results indicate that there are at least two metabolic pathways for sphingomyelin modification in cultured fibroblasts in addition to degradation by the lysosomal acid sphingomyelinase. One route is hydrolysis by a cellular sphingomyelinase. The second is the hydrolysis and/or transfer of phosphorylcholine from sphingomyelin and results in the synthesis of lecithin.

  1. Endothelial Function in Migraine With Aura – A Systematic Review

    DEFF Research Database (Denmark)

    Butt, Jawad H; Franzmann, Ulriche; Kruuse, Christina

    2015-01-01

    in migraineurs, and several studies on endothelial markers in the areas of inflammation, oxidative stress, and coagulation found increased endothelial activation in migraineurs, particularly in MA. One study, assessing cerebral endothelial function using transcranial Doppler sonography, reported lower...

  2. Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

    Science.gov (United States)

    Basso, F. G.; Pansani, T. N.; Turrioni, A. P. S.; Kurachi, C.; Bagnato, V. S.; Hebling, J.; de Souza Costa, C. A.

    2013-05-01

    Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions.

  3. Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

    International Nuclear Information System (INIS)

    Basso, F G; Pansani, T N; Turrioni, A P S; Hebling, J; De Souza Costa, C A; Kurachi, C; Bagnato, V S

    2013-01-01

    Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm −2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal–Wallis and Mann–Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions. (paper)

  4. Structural determination of intact proteins using mass spectrometry

    Science.gov (United States)

    Kruppa, Gary [San Francisco, CA; Schoeniger, Joseph S [Oakland, CA; Young, Malin M [Livermore, CA

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  5. Does human leukocyte elastase degrade intact skin elastin?

    DEFF Research Database (Denmark)

    Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes

    2012-01-01

    This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues...... and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage......, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very...

  6. SOLITARY CHEMORECEPTOR CELL SURVIVAL IS INDEPENDENT OF INTACT TRIGEMINAL INNERVATION

    Science.gov (United States)

    Gulbransen, Brian; Silver, Wayne; Finger, Tom

    2008-01-01

    Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al., 2003). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al., 2003) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. PMID:18300260

  7. Direct detection of radicals in intact soybean nodules

    DEFF Research Database (Denmark)

    Mathieu, C; Moreau, S; Frendo, P

    1998-01-01

    Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic...... differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which...... has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules...

  8. Isolation and Properties of Intact Chromoplasts from Tomato Fruits

    OpenAIRE

    Norio, Iwatsuki; Ryuichi, Moriyama; Tadashi, Asahi; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University

    1984-01-01

    Intact chromoplasts were isolated from tomato fruits at different ripening stages by Percoll density gradient centrifugation. The isolated chromoplast fractions were contaminated very little by other organelles, although the fraction from fully ripened fruits contained some mitochondria and microbodies. As the transformation of chloroplasts to chromoplasts proceeded, the density of the plastids decreased from 1.096 to 1.075g・cm^ and the decrease was related to a decrease in chlorophyll and an...

  9. Signaling hierarchy regulating human endothelial cell development

    Science.gov (United States)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  10. Endothelial dysfunction in metabolic and vascular disorders.

    Science.gov (United States)

    Polovina, Marija M; Potpara, Tatjana S

    2014-03-01

    Vascular endothelium has important regulatory functions in the cardiovascular system and a pivotal role in the maintenance of vascular health and metabolic homeostasis. It has long been recognized that endothelial dysfunction participates in the pathogenesis of atherosclerosis from early, preclinical lesions to advanced, thrombotic complications. In addition, endothelial dysfunction has been recently implicated in the development of insulin resistance and type 2 diabetes mellitus (T2DM). Considering that states of insulin resistance (eg, metabolic syndrome, impaired fasting glucose, impaired glucose tolerance, and T2DM) represent the most prevalent metabolic disorders and risk factors for atherosclerosis, it is of considerable scientific and clinical interest that both metabolic and vascular disorders have endothelial dysfunction as a common background. Importantly, endothelial dysfunction has been associated with adverse outcomes in patients with established cardiovascular disease, and a growing body of evidence indicates that endothelial dysfunction also imparts adverse prognosis in states of insulin resistance. In this review, we discuss the association of insulin resistance and T2DM with endothelial dysfunction and vascular disease, with a focus on the underlying mechanisms and prognostic implications of the endothelial dysfunction in metabolic and vascular disorders. We also address current therapeutic strategies for the improvement of endothelial dysfunction.

  11. 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

  12. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    -alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  13. A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

    Science.gov (United States)

    Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2018-01-01

    Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

  14. Global forest loss disproportionately erodes biodiversity in intact landscapes.

    Science.gov (United States)

    Betts, Matthew G; Wolf, Christopher; Ripple, William J; Phalan, Ben; Millers, Kimberley A; Duarte, Adam; Butchart, Stuart H M; Levi, Taal

    2017-07-27

    Global biodiversity loss is a critical environmental crisis, yet the lack of spatial data on biodiversity threats has hindered conservation strategies. Theory predicts that abrupt biodiversity declines are most likely to occur when habitat availability is reduced to very low levels in the landscape (10-30%). Alternatively, recent evidence indicates that biodiversity is best conserved by minimizing human intrusion into intact and relatively unfragmented landscapes. Here we use recently available forest loss data to test deforestation effects on International Union for Conservation of Nature Red List categories of extinction risk for 19,432 vertebrate species worldwide. As expected, deforestation substantially increased the odds of a species being listed as threatened, undergoing recent upgrading to a higher threat category and exhibiting declining populations. More importantly, we show that these risks were disproportionately high in relatively intact landscapes; even minimal deforestation has had severe consequences for vertebrate biodiversity. We found little support for the alternative hypothesis that forest loss is most detrimental in already fragmented landscapes. Spatial analysis revealed high-risk hot spots in Borneo, the central Amazon and the Congo Basin. In these regions, our model predicts that 121-219 species will become threatened under current rates of forest loss over the next 30 years. Given that only 17.9% of these high-risk areas are formally protected and only 8.9% have strict protection, new large-scale conservation efforts to protect intact forests are necessary to slow deforestation rates and to avert a new wave of global extinctions.

  15. Antimicrobial activity of a new intact skin antisepsis formulation.

    Science.gov (United States)

    Russo, Antonello; Viotti, Pier Luigi; Vitali, Matteo; Clementi, Massimo

    2003-04-01

    Different antiseptic formulations have shown limitations when applied to disinfecting intact skin, notably short-term tolerability and/or efficacy. The purpose of this study was optimizing a new antiseptic formulation specifically targeted at intact skin disinfection and evaluating its in vitro microbicidal activity and in vivo efficacy. The biocidal properties of the antiseptic solution containing 0.5% chloramine-T diluted in 50% isopropyl alcohol (Cloral; Eurospital SpA Trieste, Italy) were measured in vitro versus gram-positive-, gram-negative-, and acid-alcohol-resistant germs and fungi with standard suspension tests in the presence of fetal bovine serum. Virus-inhibiting activity was evaluated in vitro against human cytomegalovirus, herpes simplex virus, poliovirus, hepatitis B virus, and hepatitis C virus. Tests used different methods for the different biologic and in vitro replication capacity of these human viruses. Lastly, Cloral tolerability and skin colonization retardation efficacy after disinfection were studied in vivo. The antiseptic under review showed fast and sustained antimicrobial activity. The efficacy of Cloral against clinically important bacterial and viral pathogens and fungi was highlighted under the experimental conditions described in this article. Finally, microbial regrowth lag and no side effects were documented in vivo after disinfection of 11 volunteers. A stable chloramine-T solution in isopropyl alcohol may be suggested for intact skin antisepsis.

  16. 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)

  17. 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; ...

  18. Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway.

    Science.gov (United States)

    Tandon, Nina; Cimetta, Elisa; Villasante, Aranzazu; Kupferstein, Nicolette; Southall, Michael D; Fassih, Ali; Xie, Junxia; Sun, Ying; Vunjak-Novakovic, Gordana

    2014-01-01

    Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing. © 2013 Elsevier Inc. All rights reserved.

  19. Vascular smooth muscle modulates endothelial control of vasoreactivity via reactive oxygen species production through myoendothelial communications.

    Directory of Open Access Journals (Sweden)

    Marie Billaud

    Full Text Available BACKGROUND: Endothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC/smooth muscle cells (SMC crosstalk is far from complete. Here, we have examined the role of gap junctions and reactive oxygen species (ROS in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone. METHODOLOGY/PRINCIPAL FINDINGS: In small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses and immunofluorescent labeling evidenced connexin (Cx 37, 40 and 43 in EC and/or SMC. Functional experiments showed that the Cx-mimetic peptide targeted against Cx 37 and Cx 43 ((37,43Gap27 (1 reduced contractile and calcium responses to serotonin (5-HT simultaneously recorded in pulmonary arteries and (2 abolished the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolise superoxide anion and H(2O(2, respectively. Both Cx- and ROS-mediated effects on the responses to 5-HT were reversed by L-NAME, a NO synthase inhibitor or endothelium removal. Electronic paramagnetic resonance directly demonstrated that 5-HT-induced superoxide anion production originated from the SMC. Finally, whereas 5-HT increased NO production, it also decreased cyclic GMP content in isolated intact arteries. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that agonist-induced ROS production in SMC targeting EC via myoendothelial gap junctions reduces endothelial NO-dependent control of pulmonary vasoreactivity. Such SMC modulation of endothelial control may represent a signaling pathway controlling vasoreactivity under not only physiological but also pathological conditions that often implicate excessive ROS production.

  20. circHECTD1 promotes the silica-induced pulmonary endothelial-mesenchymal transition via HECTD1.

    Science.gov (United States)

    Fang, Shencun; Guo, Huifang; Cheng, Yusi; Zhou, Zewei; Zhang, Wei; Han, Bing; Luo, Wei; Wang, Jing; Xie, Weiping; Chao, Jie

    2018-03-14

    Excessive proliferation and migration of fibroblasts contribute to pulmonary fibrosis in silicosis, and both epithelial cells and endothelial cells participate in the accumulation of fibroblasts via the epithelial-mesenchymal transition (EMT) and the endothelial-mesenchymal transition (EndMT), respectively. A mouse endothelial cell line (MML1) was exposed to silicon dioxide (SiO 2 , 50 μg/cm 2 ), and immunofluorescence and western blot analyses were performed to evaluate levels of specific endothelial and mesenchymal markers and to elucidate the mechanisms by which SiO 2 induces the EndMT. Functional changes were evaluated by analyzing cell migration and proliferation. The mRNA and circular RNA (circRNA) levels were measured using qPCR and fluorescent in situ hybridization (FISH). Lung tissue samples from both Tie2-GFP mice exposed to SiO 2 and silicosis patients were applied to confirm the observations from in vitro experiments. Based on the results from the current study, SiO 2 increased the expression of mesenchymal markers (type I collagen (COL1A1), type III collagen (COL3A1) and alpha smooth muscle actin (α-SMA/Acta2)) and decreased the expression of endothelial markers (vascular endothelial cadherin (VE-Cad/Cdh 5) and platelet endothelial cell adhesion molecule-1 (PECAM1)), indicating the occurrence of the EndMT in response to SiO 2 exposure both in vivo and in vitro. SiO 2 concomitantly increased circHECTD1 expression, which, in turn, inhibited HECTD1 protein expression. SiO 2 -induced increases in cell proliferation, migration, and changes in marker levels were restored by either a small interfering RNA (siRNA) targeting circHECTD1 or overexpression of HECTD1 via the CRISPR/Cas9 system, confirming the involvement of the circHECTD1/HECTD1 pathway in the EndMT. Moreover, tissue samples from SiO 2 -exposed mice and silicosis patients confirmed the EndMT and change in HECTD1 expression. Our findings reveal a potentially new function for the circHECTD1/HECTD

  1. 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...

  2. Autonomic Blockade Reverses Endothelial Dysfunction in Obesity-Associated Hypertension.

    Science.gov (United States)

    Gamboa, Alfredo; Figueroa, Rocío; Paranjape, Sachin Y; Farley, Ginnie; Diedrich, Andre; Biaggioni, Italo

    2016-10-01

    Impaired nitric oxide (NO) vasodilation (endothelial dysfunction) is associated with obesity and thought to be a factor in the development of hypertension. We previously found that NO synthesis inhibition had similar pressor effects in obese hypertensives compared with healthy control during autonomic blockade, suggesting that impaired NO vasodilation is secondary to sympathetic activation. We tested this hypothesis by determining the effect of autonomic blockade (trimethaphan 4 mg/min IV) on NO-mediated vasodilation (increase in forearm blood flow to intrabrachial acetylcholine) compared with endothelial-independent vasodilation (intrabrachial sodium nitroprusside) in obese hypertensive subjects (30blood flow (from 3.9±0.7 to 5.2±1.2 mL/100 mL per minute, P=0.078). As expected, NO-mediated vasodilation was blunted on the intact day compared with NO-independent vasodilation; forearm blood flow increased from 3.6±0.6 to 10.1±1.1 with the highest dose of nitroprusside, but only from 3.7±0.4 to 7.2±0.8 mL/100 mL per minute with the highest dose of acetylcholine, Pblood flow responses to acetylcholine were restored by autonomic blockade and were no longer different to nitroprusside (from 6.2±1.1 to 11.4±1.6 mL/100 mL per minute and from 5.2±0.9 to 12.5±0.9, respectively, P=0.58). Our results support the concept that sympathetic activation contributes to the impairment in NO-mediated vasodilation seen in obesity-associated hypertension and provides further rationale to explore it as a therapeutic target. © 2016 American Heart Association, Inc.

  3. Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells

    International Nuclear Information System (INIS)

    Barchowsky, A.; Tabrizi, K.; Kent, R.S.; Whorton, A.R.

    1989-01-01

    We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51 Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H 2 O 2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells

  4. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  5. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  6. 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

  7. 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...

  8. 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...

  9. Femtosecond laser cutting of endothelial grafts: comparison of endothelial and epithelial applanation.

    Science.gov (United States)

    Bernard, Aurélien; He, Zhiguo; Gauthier, Anne Sophie; Trone, Marie Caroline; Baubeau, Emmanuel; Forest, Fabien; Dumollard, Jean Marc; Peocʼh, Michel; Thuret, Gilles; Gain, Philippe

    2015-02-01

    Stromal surface quality of endothelial lamellae cut for endothelial keratoplasty with a femtosecond laser (FSL) with epithelial applanation remains disappointing. Applanation of the endothelial side of the cornea, mounted inverted on an artificial chamber, has therefore been proposed to improve cut quality. We compared lamellar quality after FSL cutting using epithelial versus endothelial applanation. Lamellae were cut with an FSL from organ-cultured corneas. After randomization, 7 were cut with epithelial applanation and 7 with endothelial applanation. Lamellae of 50-, 75-, and 100-μm thickness were targeted. Thickness was measured by optical coherence tomography before and immediately after cutting. Viable endothelial cell density was quantified immediately after cutting using triple labeling with Hoechst/ethidium/calcein-AM coupled with image analysis with ImageJ. The stromal surface was evaluated by 9 masked observers using semiquantitative scoring of scanning electronic microscopy images. Histology of 2 samples was also analyzed before lamellar detachment. Precision (difference in target/actual thickness) and thickness regularity [coefficient of variation (CV) of 10 measurements] were significantly better with endothelial applanation (precision: 18 μm; range, 10-30; CV: 11%; range, 8-12) than with epithelial applanation (precision: 84 μm; range, 54-107; P = 0.002; CV: 24%; range, 13-47; P = 0.001). Endothelial applanation provided thinner lamellae. However, viable endothelial cell density was significantly lower after endothelial applanation (1183 cells/mm2; range, 787-1725 versus 1688 cells/mm2; range, 1288-2025; P = 0.018). FSL cutting of endothelial lamellae using endothelial applanation provides thinner more regular grafts with more predictable thickness than with conventional epithelial applanation but strongly reduces the pool of viable endothelial cells.

  10. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. (Brigham and Women' s Hospital, Boston, MA (USA))

    1989-12-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

  11. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    International Nuclear Information System (INIS)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R.

    1989-01-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake

  12. 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)

  13. 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)

  14. Resveratrol and Endothelial Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Ning Xia

    2014-10-01

    Full Text Available Nitric oxide (NO derived from the endothelial NO synthase (eNOS has antihypertensive, antithrombotic, anti-atherosclerotic and antiobesogenic properties. Resveratrol is a polyphenol phytoalexin with multiple cardiovascular and metabolic effects. Part of the beneficial effects of resveratrol are mediated by eNOS. Resveratrol stimulates NO production from eNOS by a number of mechanisms, including upregulation of eNOS expression, stimulation of eNOS enzymatic activity and reversal of eNOS uncoupling. In addition, by reducing oxidative stress, resveratrol prevents oxidative NO inactivation by superoxide thereby enhancing NO bioavailability. Molecular pathways underlying these effects of resveratrol involve SIRT1, AMPK, Nrf2 and estrogen receptors.

  15. The Pharmaceutical Device Prisma® Skin Promotes in Vitro Angiogenesis through Endothelial to Mesenchymal Transition during Skin Wound Healing.

    Science.gov (United States)

    Belvedere, Raffaella; Bizzarro, Valentina; Parente, Luca; Petrella, Francesco; Petrella, Antonello

    2017-07-25

    Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma ® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma ® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma ® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma ® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses.

  16. The Pharmaceutical Device Prisma® Skin Promotes in Vitro Angiogenesis through Endothelial to Mesenchymal Transition during Skin Wound Healing

    Directory of Open Access Journals (Sweden)

    Raffaella Belvedere

    2017-07-01

    Full Text Available Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses.

  17. 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.

  18. Feasible pickup from intact ossicular chain with floating piezoelectric microphone.

    Science.gov (United States)

    Kang, Hou-Yong; Na, Gao; Chi, Fang-Lu; Jin, Kai; Pan, Tie-Zheng; Gao, Zhen

    2012-02-22

    Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  19. Feasible pickup from intact ossicular chain with floating piezoelectric microphone

    Directory of Open Access Journals (Sweden)

    Kang Hou-Yong

    2012-02-01

    Full Text Available Abstract Objectives Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI. However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Methods Animal controlled experiment: five adult cats (eight ears were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1 the experiment group (on malleus: the FPM glued onto the handle of the malleus of the intact ossicular chains; (2 negative control group (in vivo: the FPM only hung into the tympanic cavity; (3 positive control group (Hy-M30: a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. Results The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. Conclusions It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  20. The uptake of radioactive iodine in rat intact Graafian follicles

    International Nuclear Information System (INIS)

    Lieberman, L.M.; Lieberman, G.L.; Lieberman, M.E.

    1984-01-01

    The concentration of iodine-131 in the ovaries of mammals has important implications in the use of I-131 for the diagnosis and treatment of thyroid disease in women. The authors studied the I-131 uptake in whole ovaries and in isolated Graafian follicles of sexually mature rats. Adult female Sprague-Dawley rats, in groups of 5-6 animals, were injected IP with 10-50 μCi of I-131, at 3, 12, and 24 hrs prior to the day of proestrus and killed on the day of proestrus. The thyroid gland and ovaries were removed intact and these organs, as well as eight other tissue specimens, were weighed. The large preovulatory follicles (6-9/ovary) were then isolated under a dissecting microscope and the remaining ovary weighed. All samples were counted in a gamma well counter and the % dose/g estimated. The thyroid gland showed 23.7% dose/organ at 24 hrs. Blood decreased from 1.6% dose/g at 3 hrs to 0.5% dose/g at 24 hrs with the uterus showing 1.1% dose/g and 0.4% dose/g at the same times. Ovarian tissue was 0.5, 0.1, and 0.1% dose/g at 3,12, and 24 hrs respectively, while the intact Graafian follicles had from one-tenth to one-third the concentration of the ovary at the same times. (0.05, 0.03, and 0.03% dose/g). The authors found that the intact Graafian follicle concentrates approximately one-thirtieth to one-sixteenth of the I-131 in the blood and one-tenth to one-third of the I-131 in the ovary. This suggests that there is no active uptake of I-131 in the follicle or follicular fluid

  1. Radionuclide sorption on crushed and intact granitic rock

    International Nuclear Information System (INIS)

    Eriksen, Tryggve E.; Locklund, Birgitta

    1989-05-01

    The specific surface areas and distribution ratios for sorption of 85 Sr, 137 Cs and 152 Eu were measured for crushed and intact granite rock. The experimental data can be accommodated by a sorption model encompassing sorption on outer and inner surface. It is clearly demonstrated that the time required to obtain reliable Kd-values for the sorption of strongly sorbing radionuclides like 152 Eu is very long due to solution depletion and slow diffusion into the rock. A combination of surface area measurements and batch sorption with small particles may therefore be preferable when studying strongly sorbing nuclides. (authors) (17 figs., 6 tabs.)

  2. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    International Nuclear Information System (INIS)

    Smith, D.K.; Winkler, H.H.

    1980-01-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125 I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane

  3. 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.

  4. Staphylococcus aureus extracellular adherence protein triggers TNFα release, promoting attachment to endothelial cells via protein A.

    Directory of Open Access Journals (Sweden)

    Andrew M Edwards

    Full Text Available Staphylococcus aureus is a leading cause of bacteraemia, which frequently results in complications such as infective endocarditis, osteomyelitis and exit from the bloodstream to cause metastatic abscesses. Interaction with endothelial cells is critical to these complications and several bacterial proteins have been shown to be involved. The S. aureus extracellular adhesion protein (Eap has many functions, it binds several host glyco-proteins and has both pro- and anti-inflammatory activity. Unfortunately its role in vivo has not been robustly tested to date, due to difficulties in complementing its activity in mutant strains. We previously found Eap to have pro-inflammatory activity, and here show that purified native Eap triggered TNFα release in whole human blood in a dose-dependent manner. This level of TNFα increased adhesion of S. aureus to endothelial cells 4-fold via a mechanism involving protein A on the bacterial surface and gC1qR/p33 on the endothelial cell surface. The contribution this and other Eap activities play in disease severity during bacteraemia was tested by constructing an isogenic set of strains in which the eap gene was inactivated and complemented by inserting an intact copy elsewhere on the bacterial chromosome. Using a murine bacteraemia model we found that Eap expressing strains cause a more severe infection, demonstrating its role in invasive disease.

  5. 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 ...

  6. 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.

  7. 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.

  8. The endothelial αENaC contributes to vascular endothelial function in vivo

    DEFF Research Database (Denmark)

    Tarjus, Antoine; Maase, Martina; Jeggle, Pia

    2017-01-01

    The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α β γ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldo......-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo....

  9. 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

  10. 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

  11. Metabolism of inhaled ethane and pentane by the intact rat

    International Nuclear Information System (INIS)

    Daugherty, M.S.; Luddent, T.M.; Burk, R.F.

    1986-01-01

    Measurement of exhaled ethane or pentane is a noninvasive technique for studying in vivo lipid peroxidation. Many past studies have assumed that pentane and ethane are not metabolized. Radiolabeled ( 14 C) ethane and pentane were used to study the disposition of these compounds in intact rats. Rats were placed for 8 h in a closed plexiglass chamber fitted with a system for replenishing chamber atmospheric O 2 . Evolved CO 2 was trapped by recirculating chamber air through 3 N NaOH contained in a vessel external to the chamber. Radiolabeled ethane or pentane was injected into the chamber at the start of each experiment. The percent of 14 C-activity added to the chamber recovered in the CO 2 trap, urine, and chamber air at the end of the experiment (8 h) in the [ 14 C]-ethane (n=5) and [ 14 C]-pentane (n=4) studies are presented. Results indicate that both ethane and pentane are metabolized to CO 2 in the intact rat. Possible changes in ethane and pentane metabolism must be considered if the exhalation rates of these hydrocarbons are to be used as indices of in vivo lipid peroxidation

  12. Surface plasmon resonance sensing: from purified biomolecules to intact cells.

    Science.gov (United States)

    Su, Yu-Wen; Wang, Wei

    2018-04-12

    Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

  13. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-24

    May 24, 2010 ... chronic periodontitis (CP), 31 with gingivitis (G) and 50 healthy controls. Probing depth ..... Periodontal disease in pregnancy I. Prevalence and severity. ... endothelial nitric oxide synthase gene in premenopausal women with.

  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. Dynamical Systems Approach to Endothelial Heterogeneity

    Science.gov (United States)

    Regan, Erzsébet Ravasz; Aird, William C.

    2012-01-01

    Rationale Objective Here we reexamine our current understanding of the molecular basis of endothelial heterogeneity. We introduce multistability as a new explanatory framework in vascular biology. Methods We draw on the field of non-linear dynamics to propose a dynamical systems framework for modeling multistability and its derivative properties, including robustness, memory, and plasticity. Conclusions Our perspective allows for both a conceptual and quantitative description of system-level features of endothelial regulation. PMID:22723222

  16. An ?All-laser? Endothelial Transplant

    OpenAIRE

    Rossi, Francesca; Canovetti, Annalisa; Malandrini, Alex; Lenzetti, Ivo; Pini, Roberto; Menabuoni, Luca

    2015-01-01

    The ?all laser? assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in...

  17. Endothelial microparticles: Sophisticated vesicles modulating vascular function

    Science.gov (United States)

    Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

    2015-01-01

    Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

  18. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  19. Resveratrol induces mitochondrial biogenesis in endothelial cells.

    Science.gov (United States)

    Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

    2009-07-01

    Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

  20. Effect of electrocautery on endothelial integrity of the internal thoracic artery: ultrastructural analysis with transmission electron microscopy.

    Science.gov (United States)

    Onan, Burak; Yeniterzi, Mehmet; Onan, Ismihan Selen; Ersoy, Burak; Gonca, Suheyla; Gelenli, Elif; Solakoglu, Seyhun; Bakir, Ihsan

    2014-10-01

    The internal thoracic artery (ITA) is typically harvested from the chest wall by means of conventional electrocautery. We investigated the effects of electrocautery on endothelial-cell and vessel-wall morphology at the ultrastructural level during ITA harvesting. Internal thoracic artery specimens from 20 patients who underwent elective coronary artery bypass grafting were investigated in 2 groups. The ITA grafts were sharply dissected with use of a scalpel and clips in the control group (n=10) and were harvested by means of electrocautery in the study group (n=10). Each sample was evaluated for intimal, elastic-tissue, muscular-layer, and adventitial changes. Free flow was measured intraoperatively. Light microscopic examinations were performed after hematoxylin-eosin and Masson's trichrome staining. Transmission electron microscopy was used to evaluate ultrastructural changes in the endothelial cells and vessel walls of each ITA. In the sharp-dissection group, the endothelial surfaces were lined with normal amounts of original endothelium, endothelial cells were distinctly attached to the basal lamina, cytoplasmic organelles were evident, and intercellular junctional complexes were intact. Conversely, in the electrocautery group, the morphologic integrity of endothelial cells was distorted, with some cell separations and splits, contracted cells, numerous large cytoplasmic vacuoles, and no visible cytoplasmic organelles. The subendothelial layer exhibited disintegration. Free ITA flow was higher in the sharp-dissection group (P=0.04). The integrity of endothelial cells can be better preserved when the ITA is mobilized by means of sharp dissection, rather than solely by electrocautery; we recommend a combined approach.

  1. Endothelial Mineralocorticoid Receptor Mediates Parenchymal Arteriole and Posterior Cerebral Artery Remodeling During Angiotensin II-Induced Hypertension.

    Science.gov (United States)

    Diaz-Otero, Janice M; Fisher, Courtney; Downs, Kelsey; Moss, M Elizabeth; Jaffe, Iris Z; Jackson, William F; Dorrance, Anne M

    2017-12-01

    The brain is highly susceptible to injury caused by hypertension because the increased blood pressure causes artery remodeling that can limit cerebral perfusion. Mineralocorticoid receptor (MR) antagonism prevents hypertensive cerebral artery remodeling, but the vascular cell types involved have not been defined. In the periphery, the endothelial MR mediates hypertension-induced vascular injury, but cerebral and peripheral arteries are anatomically distinct; thus, these findings cannot be extrapolated to the brain. The parenchymal arterioles determine cerebrovascular resistance. Determining the effects of hypertension and MR signaling on these arterioles could lead to a better understanding of cerebral small vessel disease. We hypothesized that endothelial MR signaling mediates inward cerebral artery remodeling and reduced cerebral perfusion during angiotensin II (AngII) hypertension. The biomechanics of the parenchymal arterioles and posterior cerebral arteries were studied in male C57Bl/6 and endothelial cell-specific MR knockout mice and their appropriate controls using pressure myography. AngII increased plasma aldosterone and decreased cerebral perfusion in C57Bl/6 and MR-intact littermates. Endothelial cell MR deletion improved cerebral perfusion in AngII-treated mice. AngII hypertension resulted in inward hypotrophic remodeling; this was prevented by MR antagonism and endothelial MR deletion. Our studies suggest that endothelial cell MR mediates hypertensive remodeling in the cerebral microcirculation and large pial arteries. AngII-induced inward remodeling of cerebral arteries and arterioles was associated with a reduction in cerebral perfusion that could worsen the outcome of stroke or contribute to vascular dementia. © 2017 American Heart Association, Inc.

  2. 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

  3. Radiation-induced adaptive response in the intact mouse

    International Nuclear Information System (INIS)

    Yonezawa, Morio

    2009-01-01

    The author and coworkers have revealed that radiation adaptive response (AR) is seen also in the bone marrow of the intact mouse, of which details are described here. First, SPF ICR mice were pre-irradiated (PI) with 0-0.1 Gy of X-ray and after 2 months, subsequently irradiated (SI) with 7.75 Gy. Survival rates at 30 days after SI were about 14% in mice with PI 0-0.025 Gy whereas 40% or more in animals with PI 0.05-0.1 Gy: bone marrow death was found significantly suppressed in this effective PI dose range. The death 2 weeks after SI was found also inhibited at PI 0.3-0.5 Gy. Second, PI doses and interval between PI and SI for acquiring the radio-resistance (RR) were studied and third, the PI 0.3-0.5 Gy with SI 8.0 Gy at 9-17 days later revealed that regional PI of the head (central nervous system) was found unnecessary for RR and of abdomen (systems of hemopoiesis, immunity and digestion), essential. Fourth, strain difference of RR was shown by the fact that RR was observed only in C57BL mouse as well, but neither in BALB/c nor C3H strain. Next, at 12 days after SI 4.25-6.75 Gy (PI 0.5 Gy at 14 days before), mouse spleen cells were subjected to colony formation analysis by counting the endogenous hemopoietic stem cells, which revealed that those cells were increased to about 5 times by PI. Suppression of SI-induced hemorrhage was found in mice with PI by the decreased fecal hemoglobin content. Finally, AR was similarly studied in p53 +/+ and its knockout C57BL mice and was not found in the latter animal, indicating the participation of p53 in AR of the intact mouse. Elucidation of AR mechanisms in the intact animal seems to require somewhat different aspect from that in cells. The results were controvertible to the general concept that radiation risk is proportional to cumulative dose, suggesting that low dose radiation differs from high dose one in biological effect. (K.T.)

  4. Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

    Science.gov (United States)

    Hubert, Astrid; Bochenek, Magdalena L; Schütz, Eva; Gogiraju, Rajinikanth; Münzel, Thomas; Schäfer, Katrin

    2017-09-01

    Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the

  5. Rosmarinic Acid Alleviates the Endothelial Dysfunction Induced by Hydrogen Peroxide in Rat Aortic Rings via Activation of AMPK

    Directory of Open Access Journals (Sweden)

    Hui Zhou

    2017-01-01

    Full Text Available Endothelial dysfunction is the key player in the development and progression of vascular events. Oxidative stress is involved in endothelial injury. Rosmarinic acid (RA is a natural polyphenol with antioxidative, antiapoptotic, and anti-inflammatory properties. The present study investigates the protective effect of RA on endothelial dysfunction induced by hydrogen peroxide (H2O2. Compared with endothelium-denuded aortic rings, the endothelium significantly alleviated the decrease of vasoconstrictive reactivity to PE and KCl induced by H2O2. H2O2 pretreatment significantly injured the vasodilative reactivity to ACh in endothelium-intact aortic rings in a concentration-dependent manner. RA individual pretreatment had no obvious effect on the vasoconstrictive reaction to PE and KCl, while its cotreatment obviously mitigated the endothelium-dependent relaxation impairments and the oxidative stress induced by H2O2. The RA cotreatment reversed the downregulation of AMPK and eNOS phosphorylation induced by H2O2 in HAEC cells. The pretreatment with the inhibitors of AMPK (compound C and eNOS (L-NAME wiped off RA’s beneficial effects. All these results demonstrated that RA attenuated the endothelial dysfunction induced by oxidative stress by activating the AMPK/eNOS pathway.

  6. Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

    Science.gov (United States)

    Langan, Stacey A; Navarro-Núñez, Leyre; Watson, Steve P; Nash, Gerard B

    2017-07-20

    Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

  7. Transport of indoleacetic acid in intact corn coleoptiles

    International Nuclear Information System (INIS)

    Parker, K.E.; Briggs, W.R.

    1990-01-01

    We have characterized the transport of [ 3 H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration

  8. Simple Genome Editing of Rodent Intact Embryos by Electroporation.

    Directory of Open Access Journals (Sweden)

    Takehito Kaneko

    Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

  9. Selective dansylation of M protein within intact influenza virions

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-12-01

    Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

  10. Selective dansylation of M protein within intact influenza virions

    International Nuclear Information System (INIS)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-01-01

    Exposure of purified influenza virions to [ 14 C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride

  11. 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.

  12. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    Science.gov (United States)

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts. Copyright © 2016 the American Physiological Society.

  13. Isolation of intact sub-dermal secretory cavities from Eucalyptus

    Directory of Open Access Journals (Sweden)

    Goodger Jason QD

    2010-09-01

    Full Text Available Abstract Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h, with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.

  14. Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

    Science.gov (United States)

    Peoples, Jessica N; Taylor, David G; Katchman, Alexander N; Ebert, Steven N

    2018-01-22

    Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh -/- ) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca 2+ ] i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca 2+ ] i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5 mM), caffeine (5 mM), and NE (100 nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I Ca,L , in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I Ca,L and that aberrant calcium signaling does not likely contribute

  15. Optimized Planning Target Volume for Intact Cervical Cancer

    International Nuclear Information System (INIS)

    Khan, Alvin; Jensen, Lindsay G.; Sun Shuai; Song, William Y.; Yashar, Catheryn M.; Mundt, Arno J.; Zhang Fuquan; Jiang, Steve B.; Mell, Loren K.

    2012-01-01

    Purpose: To model interfraction clinical target volume (CTV) variation in patients with intact cervical cancer and design a planning target volume (PTV) that minimizes normal tissue dose while maximizing CTV coverage. Methods and Materials: We analyzed 50 patients undergoing external-beam radiotherapy for intact cervical cancer using daily online cone-beam computed tomography (CBCT). The CBCTs (n = 972) for each patient were rigidly registered to the planning CT. The CTV was delineated on the planning CT (CTV 0 ) and the set of CBCTs ({CTV 1 –CTV 25 }). Manual (n = 98) and automated (n = 668) landmarks were placed over the surface of CTV 0 with reference to defined anatomic structures. Normal vectors were extended from each landmark, and the minimum length required for a given probability of encompassing CTV 1 –CTV 25 was computed. The resulting expansions were used to generate an optimized PTV. Results: The mean (SD; range) normal vector length to ensure 95% coverage was 4.3 mm (2.7 mm; 1–16 mm). The uniform expansion required to ensure 95% probability of CTV coverage was 13 mm. An anisotropic margin of 20 mm anteriorly and posteriorly and 10 mm superiorly, inferiorly, and laterally also would have ensured a 95% probability of CTV coverage. The volume of the 95% optimized PTV (1470 cm 3 ) was significantly lower than both the anisotropic PTV (2220 cm 3 ) and the uniformly expanded PTV (2110 cm 3 ) (p 0 , 5–10 mm along the interfaces of CTV 0 with the bladder and rectum, and 10–14 mm along the anterior surface of CTV 0 at the level of the uterus. Conclusion: Optimizing PTV definition according to surface landmarking resulted in a high probability of CTV coverage with reduced PTV volumes. Our results provide data justifying planning margins to use in practice and clinical trials.

  16. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  17. Reduced Ang2 expression in aging endothelial cells

    International Nuclear Information System (INIS)

    Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

    2016-01-01

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. The assessment of angiogenesis and fibroblastic stromagenesis in hyperplastic and pre-invasive breast lesions

    International Nuclear Information System (INIS)

    Pavlakis, Kitty; Messini, Irene; Vrekoussis, Thomas; Yiannou, Petros; Keramopoullos, Dimitrios; Louvrou, Niki; Liakakos, Theodoros; Stathopoulos, Efstathios N

    2008-01-01

    To investigate the changes of the neoplastic microenvironment during the different morphological alterations of hyperplastic and pre-invasive breast lesions. 78 in situ ductal carcinomas of all degrees of differentiation, 22 atypical ductal hyperplasias, 25 in situ lobular carcinomas, 18 atypical lobular hyperplasias, 32 ductal epithelial hyperplasias of usual type and 8 flat atypias were immunohistochemically investigated for the expression of vascular endothelial growth factor (VEGF), smooth muscle actin (SMA) and CD34, while microvessel density (MVD) was counted using the anti-CD31 antibody. VEGF expression was strongly correlated with MVD in all hyperplastic and pre-invasive breast lesions (p < 0.05). Stromagenesis, as characterized by an increase in SMA and a decrease in CD34 positive myofibroblasts was observed mostly around ducts harboring high grade in situ carcinoma and to a lesser extent around moderately differentiated DCIS. In these two groups of in situ carcinomas, a positive correlation between MVD and SMA (p < 0.05) was observed. On the contrary, CD34 was found to be inversely related to MVD (p < 0.05). No statistically significant changes of the stromal fibroblasts were observed in low grade DCIS neither in any of the other lesions under investigation as compared to normal mammary intra- and interlobular stroma. Angiogenesis is observed before any significant fibroblastic stromagenesis in pre-invasive breast lesions. A composite phenotype characterized by VEGF positive epithelial cells and SMA positive/CD34 negative stromal cells, is identified mostly in intermediate and high grade DCIS. These findings might imply for new therapeutic strategies using both anti-angiogenic factors and factors selectively targeting tumor stroma in order to prevent the progression of DCIS to invasive carcinoma

  3. Plasma rich in growth factors promotes dermal fibroblast proliferation, migration and biosynthetic activity.

    Science.gov (United States)

    Anitua, E; Pino, A; Orive, G

    2016-11-02

    The use of plasma rich in growth factors (PRGF) has gained importance in many medical fields due to its regenerative potential. The aim of this study is to evaluate the effects of PRGF on primary skin fibroblasts assessing cell proliferation, migration and secretion of growth factors. The age of the patients from who PRGF was prepared was also studied to determine whether it influenced the outcomes. Human dermal fibroblasts were isolated from three healthy volunteers. Using PRGF-Endoret technology, PRGF was prepared from two groups of different ages (18-35 years and 50+ years). The effects of increasing concentration of PRGF (5%, 10% and 20%) on cell proliferation and migration was evaluated. Biosynthetic behaviour of cells was also analysed measuring vascular endothelial growth factor (VEGF), transforming growth factor b1 (TGFb1) and pro-collagen type I secreted levels with or without PRGF treatment. Mean platelet enrichment reached 2.4X and 2X in 18-35 and 50+ groups respectively. A dose-dependent response was observed in proliferation assays achieving the highest levels with 20% PRGF. Migration was also promoted in cells but not in a dose-dependent manner. Cell proliferation and migration outcomes obtained with PRGF (from both groups) were significantly higher compared to non-stimulated groups (pPRGF, however, with the exception of VEGF, no statistical significances were observed between the different age groups. Results from this study concluded that PRGF is safe and effective in stimulating skin regeneration by enhancing proliferation, migration and expression of pivotal bioactive molecules involved in wound healing and haemostasis.

  4. Anti-inflammatory and apoptotic effects of the polyphenol curcumin on human fibroblast-like synoviocytes.

    Science.gov (United States)

    Kloesch, Burkhard; Becker, Tatjana; Dietersdorfer, Elisabeth; Kiener, Hans; Steiner, Guenter

    2013-02-01

    It has recently been reported that the polyphenol curcumin has pronounced anti-carcinogenic, anti-inflammatory and pro-apoptotic properties. This study investigated possible anti-inflammatory and apoptotic effects of curcumin on the human synovial fibroblast cell line MH7A, and on fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). MH7A cells and RA-FLS were stimulated either with interleukin (IL)-1β or phorbol 12-myristate 13 acetate (PMA), and treated simultaneously or sequentially with increasing concentrations of curcumin. Release of interleukin (IL)-6 and vascular endothelial growth factor (VEGF)-A was quantified by enzyme-linked immunosorbent assays (ELISAs). In MH7A cells, modulation of the transcription factor nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPKs) such as p38 and extracellular-signal regulated kinase (ERK1/2) were analysed by a reporter gene assay and Western blot, respectively. Pro-apoptotic events were monitored by Annexin-V/7-AAD based assay. Cleavage of pro-caspase-3 and -7 was checked with specific antibodies. Curcumin effectively blocked IL-1β and PMA-induced IL-6 expression both in MH7A cells and RA-FLS. VEGF-A expression could only be detected in RA-FLS and was induced by PMA, but not by IL-1β. Furthermore, curcumin inhibited activation of NF-κB and induced dephosphorylation of ERK1/2. Treatment of FLS with high concentrations of curcumin was associated with a decrease in cell viability and induction of apoptosis. The natural compound curcumin represents strong anti-inflammatory properties and induces apoptosis in FLS. This study provides an insight into possible molecular mechanisms of this substance and suggests it as a natural remedy for the treatment of chronic inflammatory diseases like RA. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. [The role of endothelial cells and endothelial precursor cells in angiogenesis].

    Science.gov (United States)

    Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

    2006-01-01

    Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

  6. Fibroblast and keratinocyte gene expression following exposure to the extracts of holy basil plant (Ocimum tenuiflorum, malabar nut plant (Justicia adhatoda, and emblic myrobalan plant (Phyllanthus emblica

    Directory of Open Access Journals (Sweden)

    Takao Someya

    2018-04-01

    Full Text Available This data article provides gene expression profiles, determined by using real-time PCR, of fibroblasts and keratinocytes treated with 0.01% and 0.001% extracts of holy basil plant (Ocimum tenuiflorum, sri lankan local name “maduruthala”, 0.1% and 0.01% extracts of malabar nut plant (Justicia adhatoda, sri lankan local name “adayhoda” and 0.003% and 0.001% extracts of emblic myrobalan plant (Phyllanthus emblica, sri lankan local name “nelli”, harvested in Sri Lanka. For fibroblasts, the dataset includes expression profiles for genes encoding hyaluronan synthase 1 (HAS1, hyaluronan synthase 2 (HAS2, hyaluronidase-1 (HYAL1, hyaluronidase-2 (HYAL2, versican, aggrecan, CD44, collagen, type I, alpha 1 (COL1A1, collagen, type III, alpha 1 (COL3A1, collagen, type VII, alpha 1 (COL7A1, matrix metalloproteinase 1 (MMP1, acid ceramidase, basic fibroblast growth factor (bFGF, fibroblast growth factor-7 (FGF7, vascular endothelial growth factor (VEGF, interleukin-1 alpha (IL-1α, cyclooxygenase-2 (cox2, transforming growth factor beta (TGF-β, and aquaporin 3 (AQP3. For keratinocytes, the expression profiles are for genes encoding HAS1, HAS2, HYAL1, HYAL2, versican, CD44, IL-1α, cox2, TGF-β, AQP3, Laminin5, collagen, type XVII, alpha 1 (COL17A1, integrin alpha-6 (ITGA6, ceramide synthase 3 (CERS3, elongation of very long chain fatty acids protein 1 (ELOVL1, elongation of very long chain fatty acids protein 4 (ELOVL4, filaggrin (FLG, transglutaminase 1 (TGM1, and keratin 1 (KRT1. The expression profiles are provided as bar graphs. Keywords: Real-time PCR, Gene expression profile, Fibroblast, Keratinocyte, Holy basil extract, Ocimum tenuiflorum, Maduruthala, Malabar nut plant extract, Justicia adhatoda, Adayhoda, Emblic myrobalan extract, Phyllanthus emblica, Nelli

  7. 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.

  8. Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

    International Nuclear Information System (INIS)

    Traber, M.G.; Thellman, C.A.; Rindler, M.J.; Kayden, H.J.

    1988-01-01

    The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 [PEG 1000]) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. [ 14 C]TPGS (synthesized from [ 14 C]PEG 1000) was taken up by Caco-2 cells but [ 14 C]PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the [ 14 C]TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule

  9. The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.

    Science.gov (United States)

    Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M

    2014-11-15

    Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.

  10. Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

    Science.gov (United States)

    Schäfer, Nicola; Lohmann, Christine; Winnik, Stephan; van Tits, Lambertus J; Miranda, Melroy X; Vergopoulos, Athanasios; Ruschitzka, Frank; Nussberger, Jürg; Berger, Stefan; Lüscher, Thomas F; Verrey, François; Matter, Christian M

    2013-12-01

    Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

  11. 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

  12. Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Yulia Shamis

    Full Text Available Human embryonic and induced pluripotent stem cells (hESC/hiPSC are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.

  13. Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

    Science.gov (United States)

    Blero, Daniel; Zhang, Jing; Pesesse, Xavier; Payrastre, Bernard; Dumont, Jacques E; Schurmans, Stéphane; Erneux, Christophe

    2005-05-01

    SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

  14. The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    van Winkelhoff Arie J

    2010-01-01

    Full Text Available Abstract Background Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS. Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains. Results To examine the role of the CPS in host-pathogen interactions we constructed an insertional isogenic P. gingivalis knockout in the epimerase-coding gene epsC that is located at the end of the CPS biosynthesis locus. This mutant was subsequently shown to be non-encapsulated. K1 capsule biosynthesis could be restored by in trans expression of an intact epsC gene. We used the epsC mutant, the W83 wild type strain and the complemented mutant to challenge human gingival fibroblasts to examine the immune response by quantification of IL-1β, IL-6 and IL-8 transcription levels. For each of the cytokines significantly higher expression levels were found when fibroblasts were challenged with the epsC mutant compared to those challenged with the W83 wild type, ranging from two times higher for IL-1β to five times higher for IL-8. Conclusions These experiments provide the first evidence that P. gingivalis CPS acts as an interface between the pathogen and the host that may reduce the host's pro-inflammatory immune response. The higher virulence of encapsulated strains may be caused by this phenomenon which enables the bacteria to evade the immune system.

  15. 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.

  16. 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

  17. Dietary phosphorus acutely impairs endothelial function.

    Science.gov (United States)

    Shuto, Emi; Taketani, Yutaka; Tanaka, Rieko; Harada, Nagakatsu; Isshiki, Masashi; Sato, Minako; Nashiki, Kunitaka; Amo, Kikuko; Yamamoto, Hironori; Higashi, Yukihito; Nakaya, Yutaka; Takeda, Eiji

    2009-07-01

    Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

  18. Transport of lipoprotein lipase across endothelial cells

    International Nuclear Information System (INIS)

    Saxena, U.; Klein, M.G.; Goldberg, I.J.

    1991-01-01

    Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, the authors studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125 I-labeled LPL ( 125 I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (1) in the medium of the upper chamber, and (2) bound to the apical cell surface. The amount of 125 I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate to the lower chamber decreased the amount of 125 I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires haparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. They postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo

  19. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  20. 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

  1. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

    Science.gov (United States)

    Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

    2017-01-01

    Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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....

  7. 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.

  8. 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

  9. Comparison of the phosphorylation events in membranes prepared from proliferating versus quiescent endothelial cells

    International Nuclear Information System (INIS)

    Kazlauskas, A.; DiColeto, P.E.

    1986-01-01

    Little is known of the intracellular events which regulate the proliferation of endothelial cells (EC). Triton-solubilized membranes from proliferating (sparse) and quiescent (confluent) EC were incubated at pH 6.5 in the presence of divalent cations and [ 32 P]ATP. Membrane proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The overall kinase activity per mg protein was slightly greater in membranes prepared from proliferating versus quiescent cells. They found four proteins labeled in sparse cells to a dramatically greater extent having the following approximate molecular masses: 180, 100, 97 and 55 kilodalton (kd). The first two phosphoproteins were phosphorylated on serine residues exclusively; the 97 kd phosphoprotein contained 39% phosphoserine (p-ser) and 61% phosphothreonine (p-thr); and the 55 kd phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr). The kinases acting on all four phosphoproteins were independent of Ca 2+ , cAMP, cGMP, or phorbol 12-myristate 13-acetate. The observed differences in phosphorylation events between sparse and confluent membranes occurred in membranes from two EC lines - pig aortic and bovine aortic - but were not apparent in membranes prepared from human foreskin fibroblasts or 3T3 cells. Sparse endothelial cells made quiescent by serum deprivation were found to resemble confluent cells in the kinase activity; therefore, the enhanced kinase activity in sparse membranes may be growth dependent

  10. Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis.

    Science.gov (United States)

    Rosmorduc, O; Wendum, D; Corpechot, C; Galy, B; Sebbagh, N; Raleigh, J; Housset, C; Poupon, R

    1999-10-01

    We tested the potential role of vascular endothelial growth factor (VEGF) and of fibroblast growth factor-2 (FGF-2) in the angiogenesis associated with experimental liver fibrogenesis induced by common bile duct ligation in Sprague-Dawley rats. In normal rats, VEGF and FGF-2 immunoreactivities were restricted to less than 3% of hepatocytes. One week after bile duct ligation, hypoxia was demonstrated by the immunodetection of pimonidazole adducts unevenly distributed throughout the lobule. After 2 weeks, hypoxia and VEGF expression were detected in >95% of hepatocytes and coexisted with an increase in periportal vascular endothelial cell proliferation, as ascertained by Ki67 immunolabeling. Subsequently, at 3 weeks the density of von Willebrand-labeled vascular section in fibrotic areas significantly increased. Semiquantitative reverse transcription polymerase chain reaction showed that VEGF(120) and VEGF(164) transcripts, that correspond to secreted isoforms, increased within 2 weeks, while VEGF(188) transcripts remained unchanged. FGF-2 mainly consisting of a 22-kd isoform, according to Western blot, was identified by immunohistochemistry in 49% and 100% of hepatocytes at 3 and 7 weeks, respectively. Our data provide evidence that in biliary-type liver fibrogenesis, angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia while FGF-2 likely contributes to the maintenance of angiogenesis at later stages.

  11. Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Mishima Satoshi

    2009-11-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Digested BLG can induce tolerance when co-administered with intact BLG in Brown Norway rats

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Barkholt, Vibeke; Madsen, Charlotte Bernhard

    the human gastro-duodenal digestion process. Four different fractions of BLG-digest was made, based on sizes of peptides or aggregates hereof. Intact BLG and the four fractions of BLG-digesta were characterized by protein chemical analyses. Brown Norway (BN) rats were immunised i.p. three times without......Background: Milk is a major constituent of small children’s diet. Milk allergy is also one of the most common allergies in small children. Prevention, treatment and general understanding of this allergy are therefore important. Methods: Intact BLG was digested in an in vitro model simulating...... the use of adjuvant with either PBS (control), 200 µg of intact BLG, 30 µg of intact BLG, 200 µg of digested BLG (with 30 µg of intact BLG), 200 µg of digested BLG, 200 µg of a fraction of large complexes or 200 µg of a fraction of small complexes (all three without intact BLG). Sera from BN rats were...

  18. Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

    Science.gov (United States)

    Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

    2017-11-01

    Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

    Directory of Open Access Journals (Sweden)

    Valerie E. Ryman

    2016-01-01

    Full Text Available Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1 metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE, can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE, is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

  20. Iron and obesity status-associated insulin resistance influence circulating fibroblast-growth factor-23 concentrations.

    Directory of Open Access Journals (Sweden)

    José Manuel Fernández-Real

    Full Text Available Fibroblast growth factor 23 (FGF-23 is known to be produced by the bone and linked to metabolic risk. We aimed to explore circulating FGF-23 in association with fatness and insulin sensitivity, atherosclerosis and bone mineral density (BMD. Circulating intact FGF-23 (iFGF-23 and C-terminal (CtFGF-23 concentrations (ELISA were measured in 133 middle aged men from the general population in association with insulin sensitivity (Cohort 1; and in association with fat mass and bone mineral density (DEXA and atherosclerosis (intima media thickness, IMT in 78 subjects (52 women with a wide range of adiposity (Cohort 2. Circulating iFGF-23 was also measured before and after weight loss. In all subjects as a whole, serum intact and C-terminal concentrations were linearly and positively associated with BMI. In cohort 1, both serum iFGF-23 and CtFGF-23 concentrations increased with insulin resistance. Serum creatinine contributed to iFGF-23 variance, while serum ferritin and insulin sensitivity (but not BMI, age or serum creatinine contributed to 17% of CtFGF-23 variance. In cohort 2, CtFGF-23 levels were higher in women vs. men, and increased with BMI, fat mass, fasting and post-load serum glucose, insulin, HOMA-IR and PTH, being negatively associated with circulating vitamin D and ferritin levels. The associations of CtFGF-23 with bone density in the radius, lumbar spine and carotid IMT were no longer significant after controlling for BMI. Weight loss led to decreased iFGF-23 concentrations. In summary, the associations of circulating FGF-23 concentration with parameters of glucose metabolism, bone density and atherosclerosis are dependent on iron and obesity status-associated insulin resistance.

  1. 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.

  2. 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

  3. [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.

  4. Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

    African Journals Online (AJOL)

    induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

  5. The role of inorganic phosphate in intact human erythrocytes

    International Nuclear Information System (INIS)

    Nishiguchi, Eiko; Umeda, Masahiro.

    1988-01-01

    The role of inorganic phosphate in intact human erythrocytes was investigated by phosphorus-31 nuclear magnetic resonance ( 31 P NMR). When erythrocytes stored for 5 weeks were incubated at 37 deg C, pH 7.4, in medium containing 2 mM adenine and 10 mM inosine, with or without 5 mM glucose, a substance of around 4 ppm, as assessed by 31 P NMR chemical shift, was detected in the mixture. However, this substance disappeared by the addition of inorganic phosphate. When erythrocytes stored for 4 weeks in acid citrate dextrose (ACD) solution were incubated with 2 mM adenine, 10 mM inosine, 5 mM glucose, 50 mM inorganic phosphate and 10 mM pyruvate at 37 deg C, pH 7.4, the 2,3-DPG level increased gradually, whereas the ATP level initially increased and then decreased. Intracellular inorganic phosphate appeared to be used for the synthesis of ATP and 2,3-DPG during the first 30 min. of the reaction. These results suggests that the inorganic phosphate accelerates glycolysis by increasing the activity of glycolytic enzymes rather than its direct involvement in synthesizing organic phosphorus compounds in stored erythrocytes. The results also suggests that the reserve energy from ATP synthesis is not sufficient for the synthesis of 2,3-DPG. (author)

  6. Basic timing abilities stay intact in patients with musician's dystonia.

    Directory of Open Access Journals (Sweden)

    M C van der Steen

    Full Text Available Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15 and a matched control group (N = 15 on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo. In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument.

  7. Basic Timing Abilities Stay Intact in Patients with Musician's Dystonia

    Science.gov (United States)

    van der Steen, M. C.; van Vugt, Floris T.; Keller, Peter E.; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  8. Intact information sampling in mesial temporal lobe epilepsy.

    Science.gov (United States)

    Zamarian, Laura; Trinka, Eugen; Kuchukhidze, Giorgi; Bodner, Thomas; Unterberger, Iris; Luef, Gerhard; Delazer, Margarete

    2015-11-01

    Previous studies have reported deficits in decision making under ambiguity for patients with mesial temporal lobe epilepsy (mTLE). It is unknown whether mTLE is also associated with alterations at a predecisional stage. This study aimed to gain insight into predecisional processing of patients with mTLE. We compared performance of patients with mTLE (n = 25) with that of healthy controls (n = 75) on the information sampling task (IST), a task assessing reflection-impulsivity and predecisional information sampling. Patients and healthy controls showed a similar performance pattern in both conditions of the IST as indicated by the amount of information gathered, the degree of uncertainty tolerated, and the number of decision errors made. They both also demonstrated a significant sensitivity to the different reward characteristics of the task. For the patient group, we found no significant effects on performance on the IST of epilepsy lateralization, abnormality side, structural abnormality (hippocampus vs. amygdala), and medication (monotherapy vs. polytherapy). Reflection processes and predecisional information sampling as tested by the IST are intact in mTLE. Patients collect as much information as healthy individuals and adapt their behavior according to the changing reward conditions. Our findings indicate that in well-defined risk situations, where memory demands are sufficiently minimized, patients with mTLE should be able to gather sufficient information, weight risks and benefits, and make advantageous decisions. (c) 2015 APA, all rights reserved).

  9. Assessment of mechanical strain in the intact plantar fascia.

    Science.gov (United States)

    Clark, Ross A; Franklyn-Miller, Andrew; Falvey, Eanna; Bryant, Adam L; Bartold, Simon; McCrory, Paul

    2009-09-01

    A method of measuring tri-axial plantar fascia strain that is minimally affected by external compressive force has not previously been reported. The purpose of this study was to assess the use of micro-strain gauges to examine strain in the different axes of the plantar fascia. Two intact limbs from a thawed, fresh-frozen cadaver were dissected, and a combination of five linear and one three-way rosette gauges were attached to the fascia of the foot and ankle. Strain was assessed during two trials, both consisting of an identical controlled, loaded dorsiflexion. An ICC analysis of the results revealed that the majority of gauge placement sites produced reliable measures (ICC>0.75). Strain mapping of the plantar fascia indicates that the majority of the strain is centrally longitudinal, which provides supportive evidence for finite element model analysis. Although micro-strain gauges do possess the limitation of calibration difficulty, they provide a repeatable measure of fascial strain and may provide benefits in situations that require tri-axial assessment or external compression.

  10. Temporal Ventriloquism Reveals Intact Audiovisual Temporal Integration in Amblyopia.

    Science.gov (United States)

    Richards, Michael D; Goltz, Herbert C; Wong, Agnes M F

    2018-02-01

    We have shown previously that amblyopia involves impaired detection of asynchrony between auditory and visual events. To distinguish whether this impairment represents a defect in temporal integration or nonintegrative multisensory processing (e.g., cross-modal matching), we used the temporal ventriloquism effect in which visual temporal order judgment (TOJ) is normally enhanced by a lagging auditory click. Participants with amblyopia (n = 9) and normally sighted controls (n = 9) performed a visual TOJ task. Pairs of clicks accompanied the two lights such that the first click preceded the first light, or second click lagged the second light by 100, 200, or 450 ms. Baseline audiovisual synchrony and visual-only conditions also were tested. Within both groups, just noticeable differences for the visual TOJ task were significantly reduced compared with baseline in the 100- and 200-ms click lag conditions. Within the amblyopia group, poorer stereo acuity and poorer visual acuity in the amblyopic eye were significantly associated with greater enhancement in visual TOJ performance in the 200-ms click lag condition. Audiovisual temporal integration is intact in amblyopia, as indicated by perceptual enhancement in the temporal ventriloquism effect. Furthermore, poorer stereo acuity and poorer visual acuity in the amblyopic eye are associated with a widened temporal binding window for the effect. These findings suggest that previously reported abnormalities in audiovisual multisensory processing may result from impaired cross-modal matching rather than a diminished capacity for temporal audiovisual integration.

  11. Evidence for an intact polysaccharide capsule in Bordetella pertussis.

    Science.gov (United States)

    Neo, YiLin; Li, Rui; Howe, Josephine; Hoo, Regina; Pant, Aakanksha; Ho, SiYing; Alonso, Sylvie

    2010-03-01

    Polysaccharide capsules contribute to the pathogenesis of many bacteria species by providing resistance against various defense mechanisms. The production of a capsule in Bordetella pertussis, the etiologic agent of whooping cough, has remained controversial; earlier studies reported this pathogen as a capsulated microorganism whereas the recent B. pertussis genome analysis revealed the presence of a truncated capsule locus. In this work, using transmission electron microscopy and immunostaining approaches, we provide a formal evidence for the presence of an intact microcapsule produced at the surface of both laboratory strain and clinical isolates of B. pertussis. In agreement with previous studies, we found that the capsule is optimally produced in avirulent phase. Unexpectedly, the presence of the capsule was also detected at the surface of virulent B. pertussis bacteria. Consistently, a substantial transcriptional activity of the capsule operon was detected in virulent phase, suggesting that the capsular polysaccharide may play a role during pertussis pathogenesis. In vitro assays indicated that the presence of the capsule does not affect B. pertussis adherence to mammalian cells and does not further protect the bacterium from phagocytosis, complement-mediated killing or antimicrobial peptide attack. Copyright 2009. Published by Elsevier SAS.

  12. Intact unconscious processing of eye contact in schizophrenia

    Directory of Open Access Journals (Sweden)

    Kiley Seymour

    2016-03-01

    Full Text Available The perception of eye gaze is crucial for social interaction, providing essential information about another person’s goals, intentions, and focus of attention. People with schizophrenia suffer a wide range of social cognitive deficits, including abnormalities in eye gaze perception. For instance, patients have shown an increased bias to misjudge averted gaze as being directed toward them. In this study we probed early unconscious mechanisms of gaze processing in schizophrenia using a technique known as continuous flash suppression. Previous research using this technique to render faces with direct and averted gaze initially invisible reveals that direct eye contact gains privileged access to conscious awareness in healthy adults. We found that patients, as with healthy control subjects, showed the same effect: faces with direct eye gaze became visible significantly faster than faces with averted gaze. This suggests that early unconscious processing of eye gaze is intact in schizophrenia and implies that any misjudgments of gaze direction must manifest at a later conscious stage of gaze processing where deficits and/or biases in attributing mental states to gaze and/or beliefs about being watched may play a role.

  13. Stress dependence of permeability of intact and fractured shale cores.

    Science.gov (United States)

    van Noort, Reinier; Yarushina, Viktoriya

    2016-04-01

    Whether a shale acts as a caprock, source rock, or reservoir, understanding fluid flow through shale is of major importance for understanding fluid flow in geological systems. Because of the low permeability of shale, flow is thought to be largely confined to fractures and similar features. In fracking operations, fractures are induced specifically to allow for hydrocarbon exploration. We have constructed an experimental setup to measure core permeabilities, using constant flow or a transient pulse. In this setup, we have measured the permeability of intact and fractured shale core samples, using either water or supercritical CO2 as the transporting fluid. Our measurements show decreasing permeability with increasing confining pressure, mainly due to time-dependent creep. Furthermore, our measurements show that for a simple splitting fracture, time-dependent creep will also eliminate any significant effect of this fracture on permeability. This effect of confinement on fracture permeability can have important implications regarding the effects of fracturing on shale permeability, and hence for operations depending on that.

  14. Research on intact marine ecosystems: a lost era.

    Science.gov (United States)

    Stachowitsch, Michael

    2003-07-01

    It is proposed that a new, fifth era should be added to the four historical phases of marine research identified by Rupert Riedl, specifically an era devoted to studying and ameliorating disturbed marine ecosystems. In an age of global environmental deterioration, many marine ecosystems and organisms are high on the list of threatened entities. This poor status prompts research that would otherwise have been unnecessary and hinders research that would normally have been conducted. I argue that research into intact marine ecosystems is becoming increasingly difficult, and that most of our future insights into marine habitats will stem from knowledge gained by examining various disfunctions of those systems rather than their functions. The new era will therefore differ from past research in its underlying aim, the range of topics studied, the selection and funding of those topics, the validity of its conclusions, and in its urgency. Sea turtles and cetaceans are cited as case studies at the organismic level, shallow-water benthic communities, including coral reefs, at the ecosystem level.

  15. Measurement of tritiated norepinephrine metabolism in intact rat brain

    International Nuclear Information System (INIS)

    Levitt, M.; Kowalik, S.; Barkai, A.I.

    1983-01-01

    A procedure for the study of NE metabolism in the intact rat brain is described. The method involves ventriculocisternal perfusion of the adult male rat with artificial CSF containing [ 3 H]NE. Radioactivity in the perfusate associated with NE and its metabolites 3,4-dihydroxymandelic acid (DOMA), 3,4-dihydroxphenylethyleneglycol (DHPG), 3-methoxy-4-hydroxymandelic acid (VMA), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and normetanephrine (NMN) is separated using high-performance liquid chromatography (HPLC). After 80 min the radioactivity in the perfusate reaches an apparent steady-state. Analysis of the steady-state samples shows higher activity in the fractions corresponding to DHPG and MHPG than in those corresponding to DOMA and VMA, confirming glycol formation as the major pathway of NE metabolism in rat brain. Pretreatment with an MAO inhibitor (tranylcypromine) results in a marked decrease in the deaminated metabolites DHPG and MHPG and a concurrent increase in NMN. The results indicate this to be a sensitive procedure for the in vivo determination of changes in NE metabolism. (Auth.)

  16. Structure of the intact ATM/Tel1 kinase

    Science.gov (United States)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  17. Genetic disruptions of Drosophila Pavlovian learning leave extinction learning intact.

    Science.gov (United States)

    Qin, H; Dubnau, J

    2010-03-01

    Individuals who experience traumatic events may develop persistent posttraumatic stress disorder. Patients with this disorder are commonly treated with exposure therapy, which has had limited long-term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short-term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive 1-day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we show that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single-gene mutants with known impact on associative learning to examine the effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning.

  18. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Ryohei Numata

    Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  19. Influence of Levamisole and Other Angiogenesis Inhibitors on Angiogenesis and Endothelial Cell Morphology in Vitro

    Energy Technology Data Exchange (ETDEWEB)

    Friis, Tina; Engel, Anne-Marie; Bendiksen, Christine D.; Larsen, Line S.; Houen, Gunnar, E-mail: gh@ssi.dk [Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen (Denmark)

    2013-06-24

    Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity

  20. Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

    International Nuclear Information System (INIS)

    Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

    1988-01-01

    DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

  1. [Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation].

    Science.gov (United States)

    Gong, Qimei; Jiang, Hongwei; Wang, Jinming; Ling, Junqi

    2014-05-01

    To investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation. DPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction. The relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

  2. The association between circulating endothelial progenitor cells and coronary collateral formation.

    Science.gov (United States)

    Tokgözoğlu, Lale; Yorgun, Hikmet; Gürses, Kadri Murat; Canpolat, Uğur; Ateş, Ahmet Hakan; Tülümen, Erol; Kaya, Ergün Barış; Aytemir, Kudret; Kabakçı, Giray; Tuncer, Murat; Oto, Ali

    2011-12-01

    We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography. Circulating CD133(+)/34(+) and CD34(+)/KDR(+) EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133(+)/34(+) (p collateral formation (p collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037). In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  3. Enhanced Vascular Endothelial Growth Factor Gene Expression in Ischaemic Skin of Critical Limb Ischaemia Patients

    Directory of Open Access Journals (Sweden)

    Silvia Bleda

    2012-01-01

    Full Text Available Objectives. To perform a quantitative analysis of the vascular endothelial growth factor (VEGF gene transcription in the skin of ischemic legs and provide information for VEGF in the pathogenesis in critical limb ischemia (CLI. Methods. Skin biopsies were obtained from 40 patients with CLI. Control samples came from 44 patients with chronic venous disease. VEGF gene expression was analysed using quantitative polymerase chain reaction. Results. Patients with CLI had higher skin VEGF expression than control group (RQ: 1.3 ± 0.1 versus 1, P=0.04. Conclusions. We found an association between ischemic skin and an elevated VEGF expression in legs from patients with CLI. These data support that the mechanism for VEGF upregulation in hypoxia conditions is intact and acts appropriately in the ischaemic limbs from patients with CLI.

  4. 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.

  5. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  6. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

    Science.gov (United States)

    Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

    2011-01-01

    Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  7. Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts

    Directory of Open Access Journals (Sweden)

    Oscar Villa

    2015-03-01

    Full Text Available Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–electrospray ionization–tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography–electrospray ionization–tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

  8. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    International Nuclear Information System (INIS)

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-01-01

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10 -9 M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  9. The assessment of angiogenesis and fibroblastic stromagenesis in hyperplastic and pre-invasive breast lesions

    Directory of Open Access Journals (Sweden)

    Louvrou Niki

    2008-04-01

    Full Text Available Abstract Background To investigate the changes of the neoplastic microenvironment during the different morphological alterations of hyperplastic and pre-invasive breast lesions. Methods 78 in situ ductal carcinomas of all degrees of differentiation, 22 atypical ductal hyperplasias, 25 in situ lobular carcinomas, 18 atypical lobular hyperplasias, 32 ductal epithelial hyperplasias of usual type and 8 flat atypias were immunohistochemically investigated for the expression of vascular endothelial growth factor (VEGF, smooth muscle actin (SMA and CD34, while microvessel density (MVD was counted using the anti-CD31 antibody. Results VEGF expression was strongly correlated with MVD in all hyperplastic and pre-invasive breast lesions (p Conclusion Angiogenesis is observed before any significant fibroblastic stromagenesis in pre-invasive breast lesions. A composite phenotype characterized by VEGF positive epithelial cells and SMA positive/CD34 negative stromal cells, is identified mostly in intermediate and high grade DCIS. These findings might imply for new therapeutic strategies using both anti-angiogenic factors and factors selectively targeting tumor stroma in order to prevent the progression of DCIS to invasive carcinoma.

  10. Application of Fourier transform infrared spectroscopy to biomolecular profiling of cultured fibroblast cells from Gaucher disease patients: A preliminary investigation.

    Science.gov (United States)

    Igci, Nasit; Sharafi, Parisa; Demiralp, Duygu Ozel; Demiralp, Cemil Ozerk; Yuce, Aysel; Emre, Serap Dokmeci

    2017-10-01

    Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks. The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method. Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm-1 wavenumbers. Each peak in the spectra was assigned to as organic biomolecules according to their chemical bond information. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis. We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis. Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its

  11. 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

  12. 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

  13. Oxidative stress induced pulmonary endothelial cell proliferation is ...

    African Journals Online (AJOL)

    Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

  14. Endothelial dysfunction – A predictor of atherosclerosis | Chhabra ...

    African Journals Online (AJOL)

    Endothelial dysfunction is a systemic disorder and a critical element in the pathogenesis of atherosclerotic diseases and its complications. Growing evidences suggest that the individual burden of currently known cardiovascular risk factors is not the only determinant of endothelial function; rather endothelial integrity ...

  15. Fibroblast Growth Factor-23 in Bed Rest and Spaceflight

    Science.gov (United States)

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

    2014-01-01

    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. Presented with an imbalanced dietary phosphorus to calcium ratio, increased secretion of FGF23 will inhibit renal phosphorus reabsorption, resulting in increased excretion and reduced circulating phosphorus. Increased intake and excretion of phosphorus is associated with increased kidney stone risk in both the terrestrial and microgravity environments. Highly processed foods and carbonated beverages are associated with higher phosphorus content. Ideally, the dietary calcium to phosphorus ratio should be at minimum 1:1. Nutritional requirements for spaceflight suggest that this ratio not be less than 0.67 (3), while the International Space Station (ISS) menu provides 1020 mg Ca and 1856 mg P, for a ratio of 0.55 (3). Subjects in NASA's bed rest studies, by design, have consumed intake ratios much closer to 1.0 (4). FGF23 also has an inhibitory influence on PTH secretion and 1(alpha)-hydroxylase, both of which are required for activating vitamin D with the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Decreased 1,25-dihydroxyvitamin D will result in decreased intestinal phosphorus absorption, and increased urinary phosphorus excretion (via decreased renal reabsorption). Should a decrease in 1

  16. Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions

    Czech Academy of Sciences Publication Activity Database

    Maxová, H.; Bačáková, Lucie; Lisá, Věra; Novotná, J.; Tomášová, H.; Vízek, M.; Herget, J.

    2010-01-01

    Roč. 59, č. 5 (2010), s. 711-719 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/08/0108; GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : hypoxia * metalloproteinases * tryptase * chymase * immunofluorescence Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.646, year: 2010

  17. Isolation of a cDNA for a Growth Factor of Vascular Endothelial Cells from Human Lung Cancer Cells: Its Identity with Insulin‐like Growth Factor II

    Science.gov (United States)

    Hagiwara, Koichi; Kobayashi, Tatsuo; Tobita, Masato; Kikyo, Nobuaki; Yazaki, Yoshio

    1995-01-01

    We have found growth‐promoting activity for vascular endothelial cells in the conditioned medium of a human lung cancer cell line, T3M‐11. Purification and characterization of the growth‐promoting activity have been carried out using ammonium sulfate precipitation and gel‐exclusion chromatography. The activity migrated as a single peak just after ribonuclease. It did not bind to a heparin affinity column. These results suggest that the activity is not a heparin‐binding growth factor (including fibroblast growth factors) or a vascular endothelial growth factor. To identify the molecule exhibiting the growth‐promoting activity, a cDNA encoding the growth factor was isolated through functional expression cloning in COS‐1 cells from a cDNA library prepared from T3M‐11 cells. The nucleotide sequence encoded by the cDNA proved to be identical with that of insulin‐like growth factor II. PMID:7730145

  18. 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.

  19. 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

  20. 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

  1. Influence of metabolism modifiers of cyclic nucleotides on contractility of right ventricle of rat heart with intact and removed endocardial endothelium

    Directory of Open Access Journals (Sweden)

    Savić Slađana

    2010-01-01

    Full Text Available Introduction. Endocardial endothelium, a natural biological barrier between circulating blood in heart ventricle and cells, creates a complex yet finely tuned balance of interactions with the immediate environment. Objective. We investigated the roles of theophylline, nonspecific phosphodiesterase inhibitor, and imidazole, an activator of phosphodiesterase on contractility of the right ventricle of rat heart, with intact and removed endocardial endothelium. Methods. Adult rats, of both sexes, type Wistar albino, were used in this experiment. All experiments were conducted on the preparations of the right ventricle using two experimental models. In the first experimental model, an endocardial endothelium (EE was preserved, and in the second model, an endocardial endothelium (-EE was removed using 1% solution Triton X-100. Results. Theophylline (1x10-2 mol/l expressed the positive inotropic effect on the heart, regardless of the presence of the endocardial endothelium. Inotropic response as multiple process can be induced by inhibition of phosphodiesterase, accumulation of cyclic nucleotides and activation of Ca2+ channels. Imidazole (2x10-3 mol/l increased the contractility of the right ventricle of the heart with EE. The modulator effect of endocardial endothelium on contractility of imidazole proved to be significant. As imidazole influenced the contractility of the right ventricle only in the presence of the endocardial endothelium, it is assumed that its effect is mediated via deliverance of endothelial mediators with positive inotropic effect. Conclusion. An intact endocardial endothelium is necessary for completion of contractile performance of the heart.

  2. Endothelial cell seeding on crosslinked collagen : Effects of crosslinking on endothelial cell proliferation and functional parameters

    NARCIS (Netherlands)

    Wissink, MJB; van Luyn, MJA; Dijk, F; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J

    Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper

  3. 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

  4. The intact capture of hypervelocity dust particles using underdense foams

    Science.gov (United States)

    Maag, Carl R.; Borg, J.; Tanner, William G.; Stevenson, T. J.; Bibring, J.-P.

    1994-01-01

    probability of survival for the impacting particle. The primary objectives of the experiment are to (1) Examine the morphology of primary and secondary hypervelocity impact craters. Primary attention will be paid to craters caused by ejecta during hypervelocity impacts of different substrates. (2) Determine the size distribution of ejecta by means of witness plates and collect fragments of ejecta from craters by means of momentum-sensitive mcropore foam. (3) Assess the directionality of the flux by means of penetration-hole alignment of thin films placed above the cells. (4) Capture intact the particles that perforated the thin film and entered the cell. Capture media consisted of both previously flight-tested micropore foams and aerogel. The foams had different latent heats of fusion and, accordingly, will capture particles over a range of momenta. Aerogel was incorporated into the cells to determine the minimum diameter than can be captured intact.

  5. Intensity modulated tangential beam irradiation of the intact breast

    International Nuclear Information System (INIS)

    Hong, L.; Hunt, M.; Chui, C.; Forster, K.; Lee, H.; Lutz, W.; Yahalom, J.; Kutcher, G.J.; McCormick, B.

    1997-01-01

    Purpose/Objective: The purpose of this study was to evaluate the potential benefits of intensity modulated tangential beams in the irradiation of the intact breast. The primary goal was to develop an intensity modulated treatment which would substantially decrease the dose to coronary arteries, lung and contralateral breast while still using a standard tangential beam arrangement. Improved target dose homogeneity, within the limits imposed by opposed fields, was also desired. Since a major goal of the study was the development of a technique which was practical for use on a large population of patients, the design of 'standard' intensity profiles analogous in function to conventional wedges was also investigated. Materials and Methods: Three dimensional treatment planning was performed using both conventional and intensity modulated tangential beams. Plans were developed for both the right and left breast for a range of patient sizes and shapes. For each patient, PTV, lung, heart, origin and peripheral branches of the coronary artery, and contralateral breast were contoured. Optimum tangential beam direction and shape were designed using Beams-Eye-View display and then used for both the conventional and intensity modulated plans. For the conventional plan, the optimum wedge combination and beam weighting were chosen based on the dose distribution in a single transverse plane through the field center. Intensity modulated plans were designed using an algorithm which allows the user to specify the prescribed, maximum and minimum acceptable doses and dose volume constraints for each organ of interest. Plans were compared using multiple dose distributions and DVHs. Results: Significant improvements in the doses to critical structures were achieved using the intensity modulated plan. Coronary artery dose decreased substantially for patients treated to the left breast. Ipsilateral lung and contralateral breast doses decreased for all patients. For one patient treated to

  6. Endothelial cell oxidative stress and signal transduction

    Directory of Open Access Journals (Sweden)

    ROCIO FONCEA

    2000-01-01

    Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

  7. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  8. ORIGINAL ARTICLE Relationship between endothelial nitric oxide ...

    African Journals Online (AJOL)

    salah

    The haplotype analysis confirmed ... hand, no consistent association was shown between the two SNPs and SBP or. DBP. ... Endothelial nitric oxide synthase gene polymorphisms and risk of MI .... type (-786T*+894G), the haplotypes ... Tests adjusted for age, BMI, diabetes, current smoking and alcohol consumption.

  9. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  10. Jagged gives endothelial tip cells an edge.

    Science.gov (United States)

    Suchting, Steven; Eichmann, Anne

    2009-06-12

    Sprouting blood vessels have tip cells that lead and stalk cells that follow. Benedito et al. (2009) now show that competition between endothelial cells for the tip position is regulated by glycosylation of Notch receptors and by the opposing actions of the Notch ligands Jagged1 and Delta-like 4.

  11. Intact suppression of increased false recognition in schizophrenia.

    Science.gov (United States)

    Weiss, Anthony P; Dodson, Chad S; Goff, Donald C; Schacter, Daniel L; Heckers, Stephan

    2002-09-01

    Recognition memory is impaired in patients with schizophrenia, as they rely largely on item familiarity, rather than conscious recollection, to make mnemonic decisions. False recognition of novel items (foils) is increased in schizophrenia and may relate to this deficit in conscious recollection. By studying pictures of the target word during encoding, healthy adults can suppress false recognition. This study examined the effect of pictorial encoding on subsequent recognition of repeated foils in patients with schizophrenia. The study included 40 patients with schizophrenia and 32 healthy comparison subjects. After incidental encoding of 60 words or pictures, subjects were tested for recognition of target items intermixed with 60 new foils. These new foils were subsequently repeated following either a two- or 24-word delay. Subjects were instructed to label these repeated foils as new and not to mistake them for old target words. Schizophrenic patients showed greater overall false recognition of repeated foils. The rate of false recognition of repeated foils was lower after picture encoding than after word encoding. Despite higher levels of false recognition of repeated new items, patients and comparison subjects demonstrated a similar degree of false recognition suppression after picture, as compared to word, encoding. Patients with schizophrenia displayed greater false recognition of repeated foils than comparison subjects, suggesting both a decrement of item- (or source-) specific recollection and a consequent reliance on familiarity in schizophrenia. Despite these deficits, presenting pictorial information at encoding allowed schizophrenic subjects to suppress false recognition to a similar degree as the comparison group, implying the intact use of a high-level cognitive strategy in this population.

  12. Mapping the World's Intact Forest Landscapes by Remote Sensing

    Directory of Open Access Journals (Sweden)

    Peter Potapov

    2008-12-01

    Full Text Available Protection of large natural forest landscapes is a highly important task to help fulfill different international strategic initiatives to protect forest biodiversity, to reduce carbon emissions from deforestation and forest degradation, and to stimulate sustainable forest management practices. This paper introduces a new approach for mapping large intact forest landscapes (IFL, defined as an unbroken expanse of natural ecosystems within areas of current forest extent, without signs of significant human activity, and having an area of at least 500 km2. We have created a global IFL map using existing fine-scale maps and a global coverage of high spatial resolution satellite imagery. We estimate the global area of IFL within the current extent of forest ecosystems (forest zone to be 13.1 million km2 or 23.5% of the forest zone. The vast majority of IFL are found in two biomes: Dense Tropical and Subtropical Forests (45.3% and Boreal Forests (43.8%. The lowest proportion of IFL is found in Temperate Broadleaf and Mixed Forests. The IFL exist in 66 of the 149 countries that together make up the forest zone. Three of them - Canada, Russia, and Brazil - contain 63.8% of the total IFL area. Of the world's IFL area, 18.9% has some form of protection, but only 9.7% is strictly protected, i.e., belongs to IUCN protected areas categories I-III. The world IFL map presented here is intended to underpin the development of a general strategy for nature conservation at the global and regional scales. It also defines a baseline for monitoring deforestation and forest degradation that is well suited for use with operational and cost-effective satellite data. All project results and IFL maps are available on a dedicated web site (http://www.intactforests.org.

  13. Preferential flow through intact soil cores: Effects of matrix head

    Energy Technology Data Exchange (ETDEWEB)

    Langner, H.W.; Gaber, H.M.; Wraith, J.M.; Huwe, B.; Inskeep, W.P.

    1999-12-01

    Continuous soil pores may act as pathways for preferential flow depending on their size and water status (filled or drained), the latter being largely controlled by the soil matrix head (h). The literature contains a wide range of proposed minimal pore sizes that may contribute to preferential flow. The objective of this study was to examine the relationship between h (and corresponding pore sizes) and preferential solute transport in a naturally structured soil. Tracer ({sup 3}H{sub 2}O and pentafluorobenzoic acid, [PFBA]) miscible displacement experiments were performed at several h values in intact soil cores (15-cm diameter, 30-cm length) using an apparatus especially suited to maintain constant h while collecting large effluent volumes. To test for the occurrence of preferential flow, observed breakthrough curves (BTCs) were evaluated for physical nonequilibrium (PNE) using a comparison between fitted local equilibrium (PNE) and PNE models. Fitting results of the observed BTCs indicated absence of PNE in all solute transport experiments at h {le} {minus}10 cm. Experiments at h {ge} {minus}5 cm consistently exhibited PNE conditions, indicating the presence of preferential flow. These results suggest that soil pores with effective radii of 150 {micro}m and smaller (water-filled at h = {minus}10 cm) do not contribute to preferential flow. Observed pore water velocities were not indicative of the presence or absence of preferential flow conditions. Continuous measurements of soil water content ({theta}) using time domain reflectometry (TDR) revealed that at h = {minus}10 cm, <2% of the soil volume had drained.

  14. 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

  15. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

    Directory of Open Access Journals (Sweden)

    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  16. Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

    Science.gov (United States)

    Taylor, R F; Price, T H; Schwartz, S M; Dale, D C

    1981-01-01

    We have developed a technique for growing endothelial monolayers on micropore filters. These monolayers demonstrate confluence by phase and electron microscopy and provide a functional barrier to passage of radiolabeled albumin. Neutrophils readily penetrate the monolayer in response to chemotaxin, whereas there is little movement in the absence of chemotaxin. This system offers unique advantages over available chemotaxis assays and may have wider applications in the study of endothelial function. Images PMID:7007441

  17. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

    Science.gov (United States)

    Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

    2008-08-01

    Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

  18. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    International Nuclear Information System (INIS)

    Lin, Ming-Chung; Chen, Chia-Ling; Yang, Tsan-Tzu; Choi, Pui-Ching; Hsing, Chung-Hsi; Lin, Chiou-Feng

    2012-01-01

    An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

  19. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ming-Chung [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan (China); Chen, Chia-Ling [Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Yang, Tsan-Tzu; Choi, Pui-Ching [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Hsing, Chung-Hsi [Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan (China); Department of Anesthesiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin@mail.ncku.edu.tw [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China)

    2012-12-01

    An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

  20. 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.

  1. 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.

  2. Endothelial microparticles: Pathogenic or passive players in endothelial dysfunction in autoimmune rheumatic diseases?

    Science.gov (United States)

    McCarthy, E M; Wilkinson, F L; Parker, B; Alexander, M Y

    2016-11-01

    Autoimmune rheumatic diseases are characterised by systemic inflammation and complex immunopathology, with an increased risk of cardiovascular disease, initiated by endothelial dysfunction in a chronic inflammatory environment. Endothelial microparticles (EMPs) are released into the circulation from activated endothelial cells and may therefore, reflect disease severity, vascular and endothelial dysfunction, that could influence disease pathogenesis via autocrine/paracrine signalling. The exact function of EMPs in rheumatic disease remains unknown, and this has initiated research to elucidate EMP composition and function, which may be determined by the mode of endothelial activation and the micro environment. To date, EMPs are thought to play a role in angiogenesis, thrombosis and inflammation by transferring specific proteins and microRNAs (miRs) to target cells. Here, we review the mechanisms underlying the generation and composition of EMPs and the clinical and experimental studies describing the involvement of EMPs in rheumatic diseases, since we have previously shown endothelial dysfunction and an elevated risk of cardiovascular disease are characteristics in systemic lupus erythematosus. We will also discuss the potential of EMPs as future biomarkers of cardiovascular risk in these diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Reduced Ang2 expression in aging endothelial cells.

    Science.gov (United States)

    Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Differences in School Behavior and Achievement between Children from Intact, Reconstituted, and Single-Parent Families.

    Science.gov (United States)

    Featherstone, Darin R.; And Others

    1992-01-01

    Analyzed differences in school behavior and achievement among students (n=530) in grades six through nine from intact, reconstituted, and single-parent families. Students from intact, two-parent families had fewer absences and tardies, higher grade point averages, and fewer negative and more positive teacher behavioral ratings than did those from…

  5. Visual Speech Fills in Both Discrimination and Identification of Non-Intact Auditory Speech in Children

    Science.gov (United States)

    Jerger, Susan; Damian, Markus F.; McAlpine, Rachel P.; Abdi, Herve

    2018-01-01

    To communicate, children must discriminate and identify speech sounds. Because visual speech plays an important role in this process, we explored how visual speech influences phoneme discrimination and identification by children. Critical items had intact visual speech (e.g. baez) coupled to non-intact (excised onsets) auditory speech (signified…

  6. The Role of Hypertriglyceridemia in the Development of Atherosclerosis and Endothelial Dysfunction

    Directory of Open Access Journals (Sweden)

    Saki Matsumoto

    2014-03-01

    Full Text Available A hereditary postprandial hypertriglyceridemic rabbit (PHT rabbit is a new dyslipidemic model showing remarkably high plasma triglycerides with only limited elevation of plasma total cholesterol. In PHT rabbits, plasma triglyceride was markedly elevated postprandially compared with healthy Japanese white (JW rabbits. In physiological experiments, the ring preparation of the thoracic aorta was suspended in an organ bath filled with modified Krebs-Henseleit solution, and the developed tension was recorded. Endothelial function was evaluated by acetylcholine-induced vasorelaxation in each preparation with intact endothelium. The acetylcholine-induced endothelium-dependent relaxation was diminished in PHT compared with JW rabbits, suggesting endothelial dysfunction in PHT rabbits. Histological examination was carried out in adipose tissue, liver and aorta. They were fixed in formaldehyde and embedded in paraffin. The tissues were sliced (4 μm and stained using hematoxylin-eosin solution. In the adipose tissue, the visceral fat accumulated, and the size of adipose cells was enlarged in PHT rabbits. The liver of the PHT rabbit was fatty and degenerated. In aorta, increased intimal thickness was observed, suggesting the progression of atherosclerosis in the PHT rabbit. This study suggests the important role of postprandial hypertriglyceridemia in atherosclerosis. By using PHT rabbits, the effects of hypertriglyceridemia on health and diseases could be evaluated precisely.

  7. The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

    Science.gov (United States)

    Giorgetti-Peraldi, S; Murdaca, J; Mas, J C; Van Obberghen, E

    2001-07-05

    Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Liver polyribosome distribution in intact and adrenalectomized rats exposed to. gamma. radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yatvin, M B; Abdel-Halim, M N [Wisconsin Univ., Madison (USA). Dept. of Radiology; Wisconsin Univ., Madison (USA). Dept. of Human Oncology)

    1978-06-01

    The mechanism(s) by which gamma radiation influences liver polyribosome distribution was studied in groups of intact and adrenalectomized male rats. A shift from light to heavy aggregates occurred in the polyribosomes of both intact and adrenalectomized rats after they were exposed to gamma rays. In irradiated adrenalectomized rats, however, the shift to heavier aggregates was not as great as that which occurred in irradiated adrenal-intact animals. Subcutaneous injection of cortisone acetate (10 mg/100 g body weight) also altered the liver polyribosome patterns of both intact and adrenalectomized rats within 8 hours of its administration. The shift which occurred following cortisone administration, however, was less striking than that seen after irradiation only. Thus, although adrenal glucocorticoids contribute to the radiation-indu ied shift in liver polyribosomes in adrenal-intact rats, other factors appear to be involved, since the shift is also obtained in adrenalectomized animals.

  13. Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

    Science.gov (United States)

    Moccia, Francesco; Bertoni, Giuseppe; Pla, Alessandra Florio; Dragoni, Silvia; Pupo, Emanuela; Merlino, Annalisa; Mancardi, Daniele; Munaron, Luca; Tanzi, Franco

    2011-09-01

    Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.

  14. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Science.gov (United States)

    Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  15. Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.

    Directory of Open Access Journals (Sweden)

    Philip E Morgan

    Full Text Available Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH. We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC were incubated with high glucose (25 mM, 24 h, 37°C, or methylglyoxal (MGO, glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C, before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05 decreased total thiols (∼35%, further experiments with MGO showed significant losses of GSH (∼40% and NADPH (∼10%; these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10% NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE formed; lower levels of N(ε-(carboxyethyllysine (CEL and N(ε-(carboxymethyllysine (CML were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.

  16. Acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress.

    Science.gov (United States)

    Carda, Ana P P; Marchi, Katia C; Rizzi, Elen; Mecawi, André S; Antunes-Rodrigues, José; Padovan, Claudia M; Tirapelli, Carlos R

    2015-01-01

    We hypothesized that acute stress would induce endothelial dysfunction. Male Wistar rats were restrained for 2 h within wire mesh. Functional and biochemical analyses were conducted 24 h after the 2-h period of restraint. Stressed rats showed decreased exploration on the open arms of an elevated-plus maze (EPM) and increased plasma corticosterone concentration. Acute restraint stress did not alter systolic blood pressure, whereas it increased the in vitro contractile response to phenylephrine and serotonin in endothelium-intact rat aortas. NG-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase, NOS, inhibitor) did not alter the contraction induced by phenylephrine in aortic rings from stressed rats. Tiron, indomethacin and SQ29548 reversed the increase in the contractile response to phenylephrine induced by restraint stress. Increased systemic and vascular oxidative stress was evident in stressed rats. Restraint stress decreased plasma and vascular nitrate/nitrite (NOx) concentration and increased aortic expression of inducible (i) NOS, but not endothelial (e) NOS. Reduced expression of cyclooxygenase (COX)-1, but not COX-2, was observed in aortas from stressed rats. Restraint stress increased thromboxane (TX)B(2) (stable TXA(2) metabolite) concentration but did not affect prostaglandin (PG)F2α concentration in the aorta. Restraint reduced superoxide dismutase (SOD) activity, whereas concentrations of hydrogen peroxide (H(2)O(2)) and reduced glutathione (GSH) were not affected. The major new finding of our study is that restraint stress increases vascular contraction by an endothelium-dependent mechanism that involves increased oxidative stress and the generation of COX-derived vasoconstrictor prostanoids. Such stress-induced endothelial dysfunction could predispose to the development of cardiovascular diseases.

  17. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  18. Effect of surface charge of immortalized mouse cerebral endothelial cell monolayer on transport of charged solutes.

    Science.gov (United States)

    Yuan, Wei; Li, Guanglei; Gil, Eun Seok; Lowe, Tao Lu; Fu, Bingmei M

    2010-04-01

    Charge carried by the surface glycocalyx layer (SGL) of the cerebral endothelium has been shown to significantly modulate the permeability of the blood-brain barrier (BBB) to charged solutes in vivo. The cultured monolayer of bEnd3, an immortalized mouse cerebral endothelial cell line, is becoming a popular in vitro BBB model due to its easy growth and maintenance of many BBB characteristics over repeated passages. To test whether the SGL of bEnd3 monolayer carries similar charge as that in the intact BBB and quantify this charge, which can be characterized by the SGL thickness (L(f)) and charge density (C(mf)), we measured the solute permeability of bEnd3 monolayer to neutral solutes and to solutes with similar size but opposite charges: negatively charged alpha-lactalbumin (-11) and positively charged ribonuclease (+3). Combining the measured permeability data with a transport model across the cell monolayer, we predicted the L(f) and the C(mf) of bEnd3 monolayer, which is approximately 160 nm and approximately 25 mEq/L, respectively. We also investigated whether orosomucoid, a plasma glycoprotein modulating the charge of the intact BBB, alters the charge of bEnd3 monolayer. We found that 1 mg/mL orosomucoid would increase SGL charge density of bEnd3 monolayer to approximately 2-fold of its control value.

  19. Intensity-modulated tangential beam irradiation of the intact breast

    International Nuclear Information System (INIS)

    Hong, L.; Hunt, M.; Chui, C.; Spirou, S.; Forster, K.; Lee, H.; Yahalom, J.; Kutcher, G.J.; McCormick, B.

    1999-01-01

    Purpose: To evaluate the potential benefits of intensity modulated tangential beams in the irradiation of the intact breast. Methods and Materials: Three-dimensional treatment planning was performed on five left and five right breasts using standard wedged and intensity modulated (IM) tangential beams. Optimal beam parameters were chosen using beams-eye-view display. For the standard plans, the optimal wedge angles were chosen based on dose distributions in the central plane calculated without inhomogeneity corrections, according to our standard protocol. Intensity-modulated plans were generated using an inverse planning algorithm and a standard set of target and critical structure optimization criteria. Plans were compared using multiple dose distributions and dose volume histograms for the planning target volume (PTV), ipsilateral lung, coronary arteries, and contralateral breast. Results: Significant improvements in the doses to critical structures were achieved using intensity modulation. Compared with a standard-wedged plan prescribed to 46 Gy, the dose from the IM plan encompassing 20% of the coronary artery region decreased by 25% (from 36 to 27 Gy) for patients treated to the left breast; the mean dose to the contralateral breast decreased by 42% (from 1.2 to 0.7 Gy); the ipsilateral lung volume receiving more than 46 Gy decreased by 30% (from 10% to 7%); the volume of surrounding soft tissue receiving more than 46 Gy decreased by 31% (from 48% to 33%). Dose homogeneity within the target volume improved greatest in the superior and inferior regions of the breast (approximately 8%), although some decrease in the medial and lateral high-dose regions (approximately 4%) was also observed. Conclusion: Intensity modulation with a standard tangential beam arrangement significantly reduces the dose to the coronary arteries, ipsilateral lung, contralateral breast, and surrounding soft tissues. Improvements in dose homogeneity throughout the target volume can also be

  20. Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

    KAUST Repository

    Malara, N.M.

    2015-03-01

    Aim: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods and results: Isolated primary fragment of different vessel types was expanded in Ham\\'s F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusion: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications. © 2015 The Authors. Published by Elsevier Ireland Ltd.

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

    Science.gov (United States)

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

    2018-01-13

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

  2. Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu.

    Science.gov (United States)

    Janardhanan, Rajiv; Kilari, Sreenivasulu; Leof, Edward B; Misra, Sanjay

    2015-01-01

    It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation. © 2016 The Author(s) Published by S. Karger AG, Basel.

  3. Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart.

    Science.gov (United States)

    Arjunan, Selvam; Reinartz, Michael; Emde, Barbara; Zanger, Klaus; Schrader, Jürgen

    2009-01-01

    The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. Arterial endothelial function measurement method and apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Jonathan S; Budinger, Thomas F

    2014-03-04

    A "relaxoscope" (100) detects the degree of arterial endothelial function. Impairment of arterial endothelial function is an early event in atherosclerosis and correlates with the major risk factors for cardiovascular disease. An artery (115), such as the brachial artery (BA) is measured for diameter before and after several minutes of either vasoconstriction or vasorelaxation. The change in arterial diameter is a measure of flow-mediated vasomodification (FMVM). The relaxoscope induces an artificial pulse (128) at a superficial radial artery (115) via a linear actuator (120). An ultrasonic Doppler stethoscope (130) detects this pulse 10-20 cm proximal to the point of pulse induction (125). The delay between pulse application and detection provides the pulse transit time (PTT). By measuring PTT before (160) and after arterial diameter change (170), FMVM may be measured based on the changes in PTT caused by changes in vessel caliber, smooth muscle tone and wall thickness.

  10. The effects of hydroxychloroquine on endothelial dysfunction.

    Science.gov (United States)

    Rahman, Rahana; Murthi, Padma; Singh, Harmeet; Gurusinghe, Seshini; Mockler, Joanne C; Lim, Rebecca; Wallace, Euan M

    2016-10-01

    Hydroxychloroquine is an anti-malarial drug which, due to its anti-inflammatory and immunomodulatory effects, is widely used for the treatment of autoimmune diseases. In a model of systemic lupus erythematosus hydroxychloroquine has been shown to exert protective endothelial effects. In this study, we aimed to investigate whether hydroxychloroquine was endothelial protective in an in vitro model of TNF-α and preeclamptic serum induced dysfunction. We showed that hydroxychloroquine significantly reduced the production of TNF-α and preeclamptic serum induced endothelin-1 (ET-1). Hydroxychloroquine also significantly mitigated TNF-α induced impairment of angiogenesis. These findings support the further assessment of hydroxychloroquine as an adjuvant therapy in preeclampsia. Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

  11. Young endothelial cells revive aging blood.

    Science.gov (United States)

    Chang, Vivian Y; Termini, Christina M; Chute, John P

    2017-11-01

    The hematopoietic system declines with age, resulting in decreased hematopoietic stem cell (HSC) self-renewal capacity, myeloid skewing, and immune cell depletion. Aging of the hematopoietic system is associated with an increased incidence of myeloid malignancies and a decline in adaptive immunity. Therefore, strategies to rejuvenate the hematopoietic system have important clinical implications. In this issue of the JCI, Poulos and colleagues demonstrate that infusions of bone marrow (BM) endothelial cells (ECs) from young mice promoted HSC self-renewal and restored immune cell content in aged mice. Additionally, delivery of young BM ECs along with HSCs following total body irradiation improved HSC engraftment and enhanced survival. These results suggest an important role for BM endothelial cells (ECs) in regulating hematopoietic aging and support further research to identify the rejuvenating factors elaborated by BM ECs that restore HSC function and the immune repertoire in aged mice.

  12. Microalbuminuria, endothelial dysfunction and cardiovascular risk

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B

    2000-01-01

    Microalbuminuria was originally considered to be an important new risk factor for diabetic nephropathy. More recently, it has been convincingly shown that microalbuminuria is also an independent risk factor for cardiovascular morbidity and mortality in Type 1 and Type 2 diabetic patients. Even...... in the non-diabetic background population, microalbuminuria is a risk factor for cardiovascular mortality. What is the link between increased loss of albumin in urine and cardiovascular disease and mortality? As microalbuminuria is apparently associated with increased universal vascular sieving of albumin...... evidence of endothelial dysfunction in patients with microalbuminuria, which may be the common link accounting for the associations mentioned above. In this context, a number of markers of endothelial cell dysfunction have been found to be increased in patients with microalbuminuria. In addition, a number...

  13. 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.

  14. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  15. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  16. Endothelial microparticles (EMP in physiology and pathology

    Directory of Open Access Journals (Sweden)

    Ewa Sierko

    2015-08-01

    Full Text Available Endothelial microparticles (EMP are released from endothelial cells (ECs in the process of activation and/or apoptosis. They harbor adhesive molecules, enzymes, receptors and cytoplasmic structures and express a wide range of various constitutive antigens, typical for ECs, at their surface. Under physiological conditions the concentration of EMP in the blood is clinically insignificant. However, it was reported that under pathological conditions EMP concentration in the blood might slightly increase and contribute to blood coagulation, angiogenesis and inflammation. It has been shown that EMP directly and indirectly contribute to the activation of blood coagulation. Endothelial microparticles directly participate in blood coagulation through their surface tissue factor (TF – a major initiator of blood coagulation. Furthermore, EMP exhibit procoagulant potential via expression of negatively charged phospholipids at their surface, which may promote assembly of coagulation enzymes (TF/VII, tenases and prothrombinase complexes, leading to thrombus formation. In addition, they provide a binding surface for coagulation factors: IXa, VIII, Va and IIa. Moreover, it is possible that EMP transfer TF from TF-bearing EMP to activated platelets and monocytes by binding them through adhesion molecules. Also, EMP express von Willebrand factor, which may facilitate platelet aggregation. Apart from their procoagulant properties, it was demonstrated that EMP may express adhesive molecules and metalloproteinases (MMP-2, MMP-9 at their surface and release growth factors, which may contribute to angiogenesis. Additionally, surface presence of C3 and C4 – components of the classical pathway – suggests pro-inflammatory properties of these structures. This article contains a summary of available data on the biology and pathophysiology of endothelial microparticles and their potential role in blood coagulation, angiogenesis and inflammation.

  17. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    Science.gov (United States)

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Targeted endothelial nanomedicine for common acute pathological conditions.

    Science.gov (United States)

    Shuvaev, Vladimir V; Brenner, Jacob S; Muzykantov, Vladimir R

    2015-12-10

    Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal

  19. 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

  20. 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

  1. 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...

  2. Endothelial cell adhesion to ion implanted polymers

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Y; Kusakabe, M [SONY Corp., Tokyo (Japan); Lee, J S; Kaibara, M; Iwaki, M; Sasabe, H [RIKEN (Inst. of Physical and Chemical Research), Saitama (Japan)

    1992-03-01

    The biocompatibility of ion implanted polymers has been studied by means of adhesion measurements of bovine aorta endothelial cells in vitro. The specimens used were polystyrene (PS) and segmented polyurethane (SPU). Na{sup +}, N{sub 2}{sup +}, O{sub 2}{sup +} and Kr{sup +} ion implantations were performed at an energy of 150 keV with fluences ranging from 1x10{sup 15} to 3x10{sup 17} ions/cm{sup 2} at room temperature. The chemical and physical structures of ion-implanted polymers have been investigated in order to analyze their tissue compatibility such as improvement of endothelial cell adhesion. The ion implanted SPU have been found to exhibit remarkably higher adhesion and spreading of endothelial cells than unimplanted specimens. By contrast, ion implanted PS demonstrated a little improvement of adhesion of cells in this assay. Results of FT-IR-ATR showed that ion implantation broke the original chemical bond to form new radicals such as OH, ....C=O, SiH and condensed rings. The results of Raman spectroscopy showed that ion implantation always produced a peak near 1500 cm{sup -1}, which indicated that these ion implanted PS and SPU had the same carbon structure. This structure is considered to bring the dramatic increase in the extent of cell adhesion and spreading to these ion implanted PS and SPU. (orig.).

  3. Extraembryonic origin of circulating endothelial cells.

    Directory of Open Access Journals (Sweden)

    Luc Pardanaud

    Full Text Available Circulating endothelial cells (CEC are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

  4. Extraembryonic origin of circulating endothelial cells.

    Science.gov (United States)

    Pardanaud, Luc; Eichmann, Anne

    2011-01-01

    Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

  5. Chlorpromazine-induced corneal endothelial phototoxicity

    International Nuclear Information System (INIS)

    Hull, D.S.; Csukas, S.; Green, K.

    1982-01-01

    Chlorpromazine, which has been used extensively for the treatment of psychiatric disorders, is known to accumulate in the posterior corneal stroma, lens, and uveal tract. Because it is a phototoxic compound, the potential exists for it to cause cellular damage after light exposure. Specular microscopic perfusion of corneal endothelial cells in darkness with 0.5 mM chlorpromazine HCl resulted in a swelling rate of 18 +/- 2 micrometer/hr, whereas corneas exposed to long-wavelength ultraviolet light for 3 min in the presence of 0.5 mM chlorpromazine swelled at 37 +/- 9 micrometer/hr (p less than 0.01). Preirradiation of 0.5 mM chlorpromazine solution with ultraviolet light for 30 min and subsequent corneal perfusion with the solution resulted in a corneal swelling rate of 45 +/- 19 micrometer/hr. Cornea endothelial cells perfused with 0.5 mM chlorpromazine that was preirradiated with ultraviolet light showed marked swelling on scanning electron microscopic examination, whereas those perfused with nonirradiated chlorpromazine were flat and showed a normal mosaic pattern. Combining either 500 U/ml catalase or 290 U/ml superoxide dismutase with chlorpromazine did not alter photoinduction of corneal swelling. The data suggest that corneal endothelial chlorpromazine phototoxicity is secondary to cytotoxic products resulting from the photodynamically induced decomposition of chlorpromazine and is not caused by hydrogen peroxide or superoxide anion generated during the phototoxic reaction

  6. Viscoelastic response of a model endothelial glycocalyx

    International Nuclear Information System (INIS)

    Nijenhuis, Nadja; Spaan, Jos A E; Mizuno, Daisuke; Schmidt, Christoph F

    2009-01-01

    Many cells cover themselves with a multifunctional polymer coat, the pericellular matrix (PCM), to mediate mechanical interactions with the environment. A particular PCM, the endothelial glycocalyx (EG), is formed by vascular endothelial cells at their luminal side, forming a mechanical interface between the flowing blood and the endothelial cell layer. The glycosaminoglycan (GAG) hyaluronan (HA) is involved in the main functions of the EG, mechanotransduction of fluid shear stress and molecular sieving. HA, due to its length, is the only GAG in the EG or any other PCM able to form an entangled network. The mechanical functions of the EG are, however, impaired when any one of its components is removed. We here used microrheology to measure the effect of the EG constituents heparan sulfate, chondroitin sulfate, whole blood plasma and albumin on the high-bandwidth mechanical properties of a HA solution. Furthermore, we probed the effect of the hyaldherin aggrecan, a constituent of the PCM of chondrocytes, and very similar to versican (present in the PCM of various cells, and possibly in the EG). We show that components directly interacting with HA (chondroitin sulfate and aggrecan) can increase the viscoelastic shear modulus of the polymer composite

  7. Corneal Endothelial Alterations in Chronic Renal Failure.

    Science.gov (United States)

    Sati, Alok; Jha, Ashok; Moulick, P S; Shankar, Sandeep; Gupta, Sandeep; Khan, M A; Dogra, Manu; Sangwan, Virender S

    2016-10-01

    To evaluate the corneal endothelial changes in patients with chronic renal failure. A total of 128 corneas of 128 subjects were studied, and 3 groups were formed. The first, the dialyzed group, composed of 32 corneas of 32 patients; the second, the nondialyzed group, composed of 34 corneas of 34 patients; and the third, the age-matched control group, composed of 64 corneas of 64 healthy subjects were examined by a specular microscope and the endothelial parameters were compared. The dialyzed group (enhanced level of toxins in the blood) was further analyzed to assess the influence of blood urea, serum creatinine, serum calcium, and serum phosphorus including the duration of dialysis on corneal endothelium. On comparing the 3 groups using analysis of variance and posthoc tests, a significant difference was found in the central corneal thickness (CCT) and endothelial cell density (CD) between the control (CCT: 506 ± 29 μm, CD: 2760 ± 304 cells/mm) and dialyzed groups (CCT: 549 ± 30 μm, CD: 2337 ± 324 cells/mm) [P chronic renal failure, more marked in patients undergoing hemodialysis and with raised blood urea level.

  8. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    Science.gov (United States)

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  9. Shape and compliance of endothelial cells after shear stress in vitro or from different aortic regions: scanning ion conductance microscopy study.

    Directory of Open Access Journals (Sweden)

    Claire M F Potter

    Full Text Available To measure the elongation and compliance of endothelial cells subjected to different patterns of shear stress in vitro, and to compare these parameters with the elongation and compliance of endothelial cells from different regions of the intact aorta.Porcine aortic endothelial cells were cultured for 6 days under static conditions or on an orbital shaker. The shaker generated a wave of medium, inducing pulsatile shear stress with a preferred orientation at the edge of the well or steadier shear stress with changing orientation at its centre. The topography and compliance of these cells and cells from the inner and outer curvature of ex vivo porcine aortic arches were measured by scanning ion conductance microscopy (SICM.Cells cultured under oriented shear stress were more elongated and less compliant than cells grown under static conditions or under shear stress with no preferred orientation. Cells from the outer curvature of the aorta were more elongated and less compliant than cells from the inner curvature.The elongation and compliance of cultured endothelial cells vary according to the pattern of applied shear stress, and are inversely correlated. A similar inverse correlation occurs in the aortic arch, with variation between regions thought to experience different haemodynamic stresses.

  10. Obstructive sleep apnoea syndrome, endothelial function and markers of endothelialization. Changes after CPAP.

    Science.gov (United States)

    Muñoz-Hernandez, Rocio; Vallejo-Vaz, Antonio J; Sanchez Armengol, Angeles; Moreno-Luna, Rafael; Caballero-Eraso, Candela; Macher, Hada C; Villar, Jose; Merino, Ana M; Castell, Javier; Capote, Francisco; Stiefel, Pablo

    2015-01-01

    This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy. Observational study, before and after CPAP therapy. We studied 30 patients with apnoea/hypopnoea index (AHI) >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process. After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, pDNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p<0.05, respectively) and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005) but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together. CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.

  11. Obstructive sleep apnoea syndrome, endothelial function and markers of endothelialization. Changes after CPAP.

    Directory of Open Access Journals (Sweden)

    Rocio Muñoz-Hernandez

    Full Text Available This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA syndrome at baseline and after three months with CPAP therapy.Observational study, before and after CPAP therapy.We studied 30 patients with apnoea/hypopnoea index (AHI >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA and microparticles (MPs were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF was determined as a marker of endothelial restoration process.After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p< 0.005 cf-DNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p<0.05, respectively and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05. These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005 but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together.CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.

  12. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    International Nuclear Information System (INIS)

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-01-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

  13. 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...

  14. 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

  15. 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,…

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.)

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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...

  10. 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

  11. Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

    Science.gov (United States)

    Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

    2013-10-29

    Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

  12. Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts

    Directory of Open Access Journals (Sweden)

    Bahar Uslu

    2015-03-01

    Full Text Available Background: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. Aims: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. Study Design: In vitro study. Methods: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group; cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group or chitosan solution at a concentration of 2.0% (Solution Group.Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1; and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2. Results: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight interc