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Sample records for cultured cardiac fibroblasts

  1. Effects of mibefradil on intracellular Ca2+ release in cultured rat cardiac fibroblasts and human platelets.

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    Eberhard, M; Miyagawa, K; Hermsmeyer, K; Erne, P

    1995-12-01

    The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 microM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 microM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1 microM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 microM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 microM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+ - nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (> or

  2. Cardiac fibroblasts regulate sympathetic nerve sprouting and neurocardiac synapse stability.

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    Céline Mias

    Full Text Available Sympathetic nervous system (SNS plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of

  3. Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

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    Mias, Céline; Coatrieux, Christelle; Denis, Colette; Genet, Gaël; Seguelas, Marie-Hélène; Laplace, Nathalie; Rouzaud-Laborde, Charlotte; Calise, Denis; Parini, Angelo; Cussac, Daniel; Pathak, Atul; Sénard, Jean-Michel; Galés, Céline

    2013-01-01

    Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic

  4. Metabolites of Hypoxic Cardiomyocytes Induce the Migration of Cardiac Fibroblasts.

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    Shi, Huairui; Zhang, Xuehong; He, Zekun; Wu, Zhiyong; Rao, Liya; Li, Yushu

    2017-01-01

    The migration of cardiac fibroblasts to the infarct region plays a major role in the repair process after myocardial necrosis or damage. However, few studies investigated whether early hypoxia in cardiomyocytes induces the migration of cardiac fibroblasts. The purpose of this study was to assess the role of metabolites of early hypoxic cardiomyocytes in the induction of cardiac fibroblast migration. Neonatal rat heart tissue was digested with a mixture of trypsin and collagenase at an appropriate ratio. Cardiomyocytes and cardiac fibroblasts were cultured via differential adhesion. The cardiomyocyte cultures were subjected to hypoxia for 2, 4, 6, 8, 10, and 12 h. The supernatants of the cardiomyocyte cultures were collected to determine the differences in cardiac fibroblast migration induced by hypoxic cardiomyocyte metabolites at various time points using a Transwell apparatus. Meanwhile, ELISA was performed to measure TNF-α, IL-1β and TGF-β expression levels in the cardiomyocyte metabolites at various time points. The metabolites of hypoxic cardiomyocytes significantly induced the migration of cardiac fibroblasts. The induction of cardiac fibroblast migration was significantly enhanced by cardiomyocyte metabolites in comparison to the control after 2, 4, and 6 h of hypoxia, and the effect was most significant after 2 h. The expression levels of TNF-α, IL-1β, IL-6, and TGF-β were substantially increased in the metabolites of cardiomyocytes, and neutralization with anti-TNF-α and anti-IL-1β antibodies markedly reduced the induction of cardiac fibroblast migration by the metabolites of hypoxic cardiomyocytes. The metabolites of early hypoxic cardiomyocytes can induce the migration of cardiac fibroblasts, and TNF-α and IL-1β may act as the initial chemotactic inducers. © 2017 The Author(s) Published by S. Karger AG, Basel.

  5. Cardiac fibroblast in development and wound healing.

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    Deb, Arjun; Ubil, Eric

    2014-05-01

    Cardiac fibroblasts are the most abundant cell type in the mammalian heart and comprise approximately two-thirds of the total number of cardiac cell types. During development, epicardial cells undergo epithelial-mesenchymal-transition to generate cardiac fibroblasts that subsequently migrate into the developing myocardium to become resident cardiac fibroblasts. Fibroblasts form a structural scaffold for the attachment of cardiac cell types during development, express growth factors and cytokines and regulate proliferation of embryonic cardiomyocytes. In post natal life, cardiac fibroblasts play a critical role in orchestrating an injury response. Fibroblast activation and proliferation early after cardiac injury are critical for maintaining cardiac integrity and function, while the persistence of fibroblasts long after injury leads to chronic scarring and adverse ventricular remodeling. In this review, we discuss the physiologic function of the fibroblast during cardiac development and wound healing, molecular mediators of activation that could be possible targets for drug development for fibrosis and finally the use of reprogramming technologies for reversing scar. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Transplantation of 5-azacytidine treated cardiac fibroblasts improves cardiac function of infarct hearts in rats

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    TANG Cheng-chun; MA Gan-shan; CHEN Ji-yuan

    2010-01-01

    Background Cellular cardiomyoplasty by transplantation of various cell types has been investigated as potential treatments for the improvement of cardiac function after myocardial injury. A major barrier for the clinical application of cell transplantation is obtaining sufficiently large quantities of suitable cells. AIIogeneic cellular cardiomyoplasty may provide an alternative source of abundant, transplantable, myogenic cells by in vitro manipulation of cardiac fibroblasts using chemicals including 5-azacytidine. This study evaluated cardiomyogenic differentiation of cardiac fibroblasts, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function.Methods Primary cardiac fibroblasts from neonatal rats were treated with 5-azacytidine (10 μmol/L) or control.Treatment of 5-azacytidine caused myogenic differentiation of cultured cardiac fibroblasts, as defined by elongation and fusion into multinucleated myotubes with sarcomeric structures as identified by electron microscopy, and positive immunostaining for cardiac specific proteins, troponin I and β-myosin heavy chain (β-MHC) and the gap junction protein connexin 43. The myogenic cells (1.0x106) were transplanted into the infarcted myocardium 2 weeks after coronary artery occlusion.Results By 1 month after transplantation, the converted fibroblasts gave rise to a cluster of cardiac-like muscle cells that in the hearts occupied a large part of the scar with positive immunostaining for the myogenic proteins troponin I and β-MHC. Engrafted cells also expressed the gap junction protein connexin 43 in a disorganized manner. There was no positive staining in the control hearts treated with injections of culture medium. Heart function was evaluated at 6 weeks after myocardial injury with echocardiographic and hemodynamic measurements. Improvement in cardiac function was seen in the hearts transplanted with the 5-azacytidine-treated cardiac fibroblasts which was absent in the

  7. Isolation of cardiac myocytes and fibroblasts from neonatal rat pups.

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    Golden, Honey B; Gollapudi, Deepika; Gerilechaogetu, Fnu; Li, Jieli; Cristales, Ricardo J; Peng, Xu; Dostal, David E

    2012-01-01

    Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FBs) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Both cell types are relatively easy to culture as monolayers and can be manipulated using molecular and pharmacological tools. Because NRVM cease to proliferate after birth, and FBs undergo phenotypic changes and senescence after a few passages in tissue culture, primary cultures of both cell types are required for experiments. Below we describe methods that provide good cell yield and viability of primary cultures of NRVM and FBs from 0 to 3-day-old neonatal rat pups.

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

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    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.

  9. Establishing Primary Adult Fibroblast Cultures From Rodents

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    Seluanov, Andrei; Vaidya, Amita; Gorbunova, Vera

    2010-01-01

    The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel. PMID:20972406

  10. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: Specific effect of transforming growth factor. beta

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    Eghbali, M.; Tomek, R.; Woods, C.; Bhambi, B. (Univ. of Chicago, IL (United States))

    1991-02-01

    Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-{beta}{sub 1}, (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-{beta}{sub 1} became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-{beta}{sub 1}, cell proliferation (as measured by ({sup 3}H)thymidine incorporation) was moderately increased. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-{beta}{sub 1} in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. The findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-{beta}{sub 1} seems to be a specific inducer of such conversion.

  11. Aging Impairs the Proliferative Capacity of Cardiospheres, Cardiac Progenitor Cells and Cardiac Fibroblasts: Implications for Cell Therapy

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    Jianqin Ye

    2013-09-01

    Full Text Available Introduction: Cardiospheres (CS are self-assembling clusters of cells that can be grown from cardiac tissue. They contain a heterogeneous cell population that includes cardiac progenitor cells (CPCs and cardiac fibroblasts. CS and CPCs have been shown to improve cardiac function after myocardial infarction (MI in experimental models and are now being studied in clinical trials. The effects of aging on the proliferative capacity of CS and CPCs, and the paracrine signaling between cell types, remain incompletely understood. Methods and Results: We compared the growth of CS from young and aging murine hearts at baseline and following MI. The number of CS from young and aging hearts was similar at baseline. However, after MI, young hearts had a dramatic increase in the number of CS that grew, but this proliferative response to MI was virtually abolished in the aging heart. Further, the proportion of cells within the CS that were CPCs (defined as Sca-1(stem cell antigen-1+/CD45− was significantly lower in aging hearts than young hearts. Thus the number of available CPCs after culture from aging hearts was substantially lower than from young hearts. Cardiac fibroblasts from aging hearts proliferated more slowly in culture than those from young hearts. We then investigated the interaction between aging cardiac fibroblasts and CPCs. We found no significant paracrine effects on proliferation between these cell types, suggesting the impaired proliferation is a cell-autonomous problem. Conclusions: Aging hearts generate fewer CPCs, and aging CPCs have significantly reduced proliferative potential following MI. Aging cardiac fibroblasts also have reduced proliferative capacity, but these appear to be cell-autonomous problems, not caused by paracrine signaling between cell types.

  12. Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions

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    Hiroyuki Yamakawa

    2015-12-01

    Full Text Available Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF 2, FGF10, and vascular endothelial growth factor (VEGF, termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming.

  13. In Vitro Effects of Pirfenidone on Cardiac Fibroblasts: Proliferation, Myofibroblast Differentiation, Migration and Cytokine Secretion

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    Qiang Shi; Xiaoyan Liu; Yuanyuan Bai; Chuanjue Cui; Jun Li; Yishi Li; Shengshou Hu; Yingjie Wei

    2011-01-01

    Cardiac fibroblasts (CFs) are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remode...

  14. Metformin Inhibits Angiotensin II-Induced Differentiation of Cardiac Fibroblasts into Myofibroblasts

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    Jian Bai; Na Zhang; Ying Hua; Bingjian Wang; Lin Ling; Albert Ferro; Biao Xu

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibr...

  15. EPAC expression and function in cardiac fibroblasts and myofibroblasts

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    Olmedo, Ivonne; Muñoz, Claudia; Guzmán, Nancy; Catalán, Mabel; Vivar, Raúl; Ayala, Pedro; Humeres, Claudio; Aránguiz, Pablo [Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); García, Lorena [Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Velarde, Victoria [Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile (Chile); Díaz-Araya, Guillermo, E-mail: gadiaz@ciq.uchile.cl [Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile)

    2013-10-15

    In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. Method: Rat neonatal CF and CMF were treated with TGF-β1 at different times and concentrations. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; and collagen expression was determined by western blot. Results: TGF-β1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. Conclusion: TGF-β1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling. - Highlights: • TGF-β1 regulates EPAC-1 expression in cardiac fibroblast and myofibroblast. • Rap-1GTP levels are higher in cardiac myofibroblast than fibroblast. • EPAC-1 controls adhesion, migration and collagen synthesis in cardiac

  16. The Living Scar – Cardiac Fibroblasts and the Injured Heart

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    Rog-Zielinska, Eva A; Norris, Russell A; Kohl, Peter; Markwald, Roger

    2015-01-01

    Cardiac scars, often perceived as “dead” tissue, are very much alive, with heterocellular activity ensuring the maintenance of structural and mechanical integrity following heart injury. To form a scar, non-myocytes such as fibroblasts, proliferate and are recruited from intra- and extra-cardiac sources. Fibroblasts perform important autocrine and paracrine signalling functions. They also establish mechanical and, as is increasingly evident, electrical junctions with other cells. While fibroblasts were previously thought to act simply as electrical insulators, they may be electrically connected among themselves and, under certain circumstances, to other cells, including cardiomyocytes. A better understanding of these interactions will help target scar structure and function and facilitate the development of novel therapies aimed at modifying scar properties for patient benefit. This review explores available insight and recent concepts on fibroblast integration in the heart, and highlights potential avenues for harnessing their roles to optimise scar function following heart injury such as infarction, and therapeutic interventions such as ablation. PMID:26776094

  17. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures

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    Parameswaran, Sreejit; Sharma, Rajendra K.

    2016-01-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  18. A critical role of cardiac fibroblast-derived exosomes in activating renin angiotensin system in cardiomyocytes.

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    Lyu, Linmao; Wang, Hui; Li, Bin; Qin, Qingyun; Qi, Lei; Nagarkatti, Mitzi; Nagarkatti, Prakash; Janicki, Joseph S; Wang, Xing Li; Cui, Taixing

    2015-12-01

    Chronic activation of the myocardial renin angiotensin system (RAS) elevates the local level of angiotensin II (Ang II) thereby inducing pathological cardiac hypertrophy, which contributes to heart failure. However, the precise underlying mechanisms have not been fully delineated. Herein we report a novel paracrine mechanism between cardiac fibroblasts (CF)s and cardiomyocytes whereby Ang II induces pathological cardiac hypertrophy. In cultured CFs, Ang II treatment enhanced exosome release via the activation of Ang II receptor types 1 (AT1R) and 2 (AT2R), whereas lipopolysaccharide, insulin, endothelin (ET)-1, transforming growth factor beta (TGFβ)1 or hydrogen peroxide did not. The CF-derived exosomes upregulated the expression of renin, angiotensinogen, AT1R, and AT2R, downregulated angiotensin-converting enzyme 2, and enhanced Ang II production in cultured cardiomyocytes. In addition, the CF exosome-induced cardiomyocyte hypertrophy was blocked by both AT1R and AT2R antagonists. Exosome inhibitors, GW4869 and dimethyl amiloride (DMA), inhibited CF-induced cardiomyocyte hypertrophy with little effect on Ang II-induced cardiomyocyte hypertrophy. Mechanistically, CF exosomes upregulated RAS in cardiomyocytes via the activation of mitogen-activated protein kinases (MAPKs) and Akt. Finally, Ang II-induced exosome release from cardiac fibroblasts and pathological cardiac hypertrophy were dramatically inhibited by GW4869 and DMA in mice. These findings demonstrate that Ang II stimulates CFs to release exosomes, which in turn increase Ang II production and its receptor expression in cardiomyocytes, thereby intensifying Ang II-induced pathological cardiac hypertrophy. Accordingly, specific targeting of Ang II-induced exosome release from CFs may serve as a novel therapeutic approach to treat cardiac pathological hypertrophy and heart failure.

  19. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

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    Ruvinov Emil

    2008-11-01

    Full Text Available Abstract Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO would improve tissue repair in rat after myocardial infarction (MI. Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat.

  20. Expression of fibulin-6 in failing hearts and its role for cardiac fibroblast migration.

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    Chowdhury, Arpita; Herzog, Christine; Hasselbach, Lisa; Khouzani, Houra Loghmani; Zhang, Jinli; Hammerschmidt, Matthias; Rudat, Carsten; Kispert, Andreas; Gaestel, Matthias; Menon, Manoj B; Tudorache, Igor; Hilfiker-Kleiner, Denise; Mühlfeld, Christian; Schmitto, Jan Dieter; Müller, Martin; Theilmeier, Gregor

    2014-09-01

    The cardiac extracellular matrix (ECM) undergoes a dynamic transition following myocardial infarction. Fibulin-6 is expressed in cell junctions particularly in tissues subjected to significant mechanical stress. Fibulin-6 deficiency results in defective cell migration in nematodes and early embryonic lethality in mice. The role of fibulin-6 in healthy and failing myocardium is unknown. We have examined the expression and distribution pattern of fibulin-6 during myocardial remodelling (MR) and detailed its effect on the migratory function of cardiac fibroblasts (CFs) in response to TGF-β1. In healthy murine myocardium, fibulin-6 expression is largely confined to larger coronary arteries. It is induced during the early and the late phase of remodelling after infarction in murine hearts predominantly in the scar-muscle junction. Similar results are obtained in human ischaemic cardiomyopathy. Fibulin-6 is mostly expressed in close vicinity to vimentin-positive cells and is also abundantly expressed in vitro in cultured neonatal CF. TGF-β1 does not induce smooth muscle actin in fibroblasts deficient of fibulin-6, which also compromised their migration. Cells that had migrated expressed more fibulin-6 compared with stationary cells. Plated on fibulin-6-depleted matrix, stress fibre induction in fibroblast in response to TGF-β1 was impaired. In ex vivo explant cultures from post-infarct myocardium, the number of emigrating fibroblasts was also significantly reduced by fibulin-6 siRNA knockdown. Fibulin-6, a fibroblast-released ECM protein, may play an important role during MR by imparting an effect on CF migration in close and complementary interplay with TGF-β1 signalling. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  1. Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.

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    Castaldo, Clotilde; Di Meglio, Franca; Miraglia, Rita; Sacco, Anna Maria; Romano, Veronica; Bancone, Ciro; Della Corte, Alessandro; Montagnani, Stefania; Nurzynska, Daria

    2013-01-01

    Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  2. Cardiac Fibroblast-Derived Extracellular Matrix (Biomatrix as a Model for the Studies of Cardiac Primitive Cell Biological Properties in Normal and Pathological Adult Human Heart

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    Clotilde Castaldo

    2013-01-01

    Full Text Available Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix, composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  3. In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion.

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    Qiang Shi

    Full Text Available Cardiac fibroblasts (CFs are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.

  4. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  5. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Bai

    Full Text Available Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM in the presence or absence of metformin (10-200 µM. Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  6. Comparative Analysis of Telomerase Activity in CD117+CD34+ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yuan Li; Shan-Shan Lu; Ting Xu; Hong-Qi Zhang; Hua Li

    2015-01-01

    Background:This study characterized the cardiac telocyte (TC) population both in vivo and in vitro,and investigated its telomerase activity related to mitosis.Methods:Using transmission electron microscopy and a phase contrast microscope,the typical morphological features of cardiac TCs were observed;by targeting the cell surface proteins CD 1 17 and CD34,CD 117+CD34+ cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture.Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8.Under this conditioned medium,the process of cell division was captured,and the telomerase activity ofCD 117+CD34+ cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs),cardiac fibroblasts (CFBs),cardiomyocytes (CMs).Results:Cardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms).In addition,64% of the primary cultured cardiac TCs were composed of CD 117+CD34+ cardiac TCs;which was verified by immunofluorescence.In a live cell imaging system,CD 117+CD34+ cardiac TCs were observed to enter into cell division in a short time,followed by an significant invagination forming across the middle of the cell body.Using a real-time quantitative telomeric-repeat amplification assay,the telomerase concentration in CD117+CD34+ cardiac TCs was obviously lower than in BMSCs and CFBs,and significantly higher than in CMs.Conclusions:Cardiac TCs represent a unique cell population and CD117+CD34+ cardiac TCs have relative low telomerase activity that differs from BMSCs,CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis.

  7. Comparative Analysis of Telomerase Activity in CD117+CD34+ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

    Science.gov (United States)

    Li, Yuan-Yuan; Lu, Shan-Shan; Xu, Ting; Zhang, Hong-Qi; Li, Hua

    2015-01-01

    Background: This study characterized the cardiac telocyte (TC) population both in vivo and in vitro, and investigated its telomerase activity related to mitosis. Methods: Using transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD117 and CD34, CD117+CD34+ cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117+CD34+ cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs), cardiac fibroblasts (CFBs), cardiomyocytes (CMs). Results: Cardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms). In addition, 64% of the primary cultured cardiac TCs were composed of CD117+CD34+ cardiac TCs; which was verified by immunofluorescence. In a live cell imaging system, CD117+CD34+ cardiac TCs were observed to enter into cell division in a short time, followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117+CD34+ cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs. Conclusions: Cardiac TCs represent a unique cell population and CD117+CD34+ cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis. PMID:26168836

  8. Cardiac fibroblast-dependent extracellular matrix accumulation is associated with diastolic stiffness in type 2 diabetes.

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    Kirk R Hutchinson

    Full Text Available Cardiovascular complications are a leading cause of death in patients with type 2 diabetes mellitus (T2DM. Diastolic dysfunction is one of the earliest manifestations of diabetes-induced changes in left ventricular (LV function, and results from a reduced rate of relaxation and increased stiffness. The mechanisms responsible for increased stiffness are not completely understood. Chronic hyperglycemia, advanced glycation endproducts (AGEs, and increased levels of proinflammatory and profibrotic cytokines are molecular pathways known to be involved in regulating extracellular matrix (ECM synthesis and accumulation resulting in increased LV diastolic stiffness. Experiments were conducted using a genetically-induced mouse model of T2DM generated by a point mutation in the leptin receptor resulting in nonfunctional leptin receptors (db/db murine model. This study correlated changes in LV ECM and stiffness with alterations in basal activation of signaling cascades and expression of profibrotic markers within primary cultures of cardiac fibroblasts from diabetic (db/db mice with nondiabetic (db/wt littermates as controls. Primary cultures of cardiac fibrobroblasts were maintained in 25 mM glucose (hyperglycemic-HG; diabetic db/db media or 5 mM glucose (normoglycemic-NG, nondiabetic db/wt media. The cells then underwent a 24-hour exposure to their opposite (NG; diabetic db/db media or 5 mM glucose (HG, nondiabetic db/wt media. Protein analysis demonstrated significantly increased expression of type I collagen, TIMP-2, TGF-β, PAI-1 and RAGE in diabetic db/db cells as compared to nondiabetic db/wt, independent of glucose media concentration. This pattern of protein expression was associated with increased LV collagen accumulation, myocardial stiffness and LV diastolic dysfunction. Isolated diabetic db/db fibroblasts were phenotypically distinct from nondiabetic db/wt fibroblasts and exhibited a profibrotic phenotype in normoglycemic conditions.

  9. Cardiac Fibroblast-Dependent Extracellular Matrix Accumulation Is Associated with Diastolic Stiffness in Type 2 Diabetes

    Science.gov (United States)

    Hutchinson, Kirk R.; Lord, C. Kevin; West, T. Aaron; Stewart, James A.

    2013-01-01

    Cardiovascular complications are a leading cause of death in patients with type 2 diabetes mellitus (T2DM). Diastolic dysfunction is one of the earliest manifestations of diabetes-induced changes in left ventricular (LV) function, and results from a reduced rate of relaxation and increased stiffness. The mechanisms responsible for increased stiffness are not completely understood. Chronic hyperglycemia, advanced glycation endproducts (AGEs), and increased levels of proinflammatory and profibrotic cytokines are molecular pathways known to be involved in regulating extracellular matrix (ECM) synthesis and accumulation resulting in increased LV diastolic stiffness. Experiments were conducted using a genetically-induced mouse model of T2DM generated by a point mutation in the leptin receptor resulting in nonfunctional leptin receptors (db/db murine model). This study correlated changes in LV ECM and stiffness with alterations in basal activation of signaling cascades and expression of profibrotic markers within primary cultures of cardiac fibroblasts from diabetic (db/db) mice with nondiabetic (db/wt) littermates as controls. Primary cultures of cardiac fibrobroblasts were maintained in 25 mM glucose (hyperglycemic-HG; diabetic db/db) media or 5 mM glucose (normoglycemic-NG, nondiabetic db/wt) media. The cells then underwent a 24-hour exposure to their opposite (NG; diabetic db/db) media or 5 mM glucose (HG, nondiabetic db/wt) media. Protein analysis demonstrated significantly increased expression of type I collagen, TIMP-2, TGF-β, PAI-1 and RAGE in diabetic db/db cells as compared to nondiabetic db/wt, independent of glucose media concentration. This pattern of protein expression was associated with increased LV collagen accumulation, myocardial stiffness and LV diastolic dysfunction. Isolated diabetic db/db fibroblasts were phenotypically distinct from nondiabetic db/wt fibroblasts and exhibited a profibrotic phenotype in normoglycemic conditions. PMID:23991045

  10. Effects of Benazepril and Losartan on Proliferation and Collagen Synthe sis of Cultured Cardiac Fibroblasts from SHR%Benazepril和Losartan对培养SHR心肌成纤维细胞增殖及合成胶原影响

    Institute of Scientific and Technical Information of China (English)

    谢良地; 陈水龙; 许昌声; 王华军; 吴可贵

    2001-01-01

    Objective To investigate the effects of angiotensinⅡ(AngⅡ), Benazepril(Bena) and Losartan on proliferation and collagen synthesis of cultured rat cardiac fibroblasts(CFb) derived from SHR (CFbSHR) and WKY (CFbWKY). Methods CFb derived from 12-week-old SHR and WKY was cultured by outgrowth of tissue block. Cell proliferation of CFb was determined by direct cell counting .3H-Proline incorporation of CFb was measured after incubation with AngⅡ,Bena and Losartan. Results In serum free(0.4%FCS) medium, cell number of CFb derived from SHR and WKY were not influenced by AngⅡ(10-10mol~10-6mol)and Bena(10-9mol~10-5mol). Increased 3H-proline incorporation wa s induced by AngⅡ in a concentration dependent manner. Benazepril caused an decrease in 3H-Proline incorporation in CFb derived from SHR and WKY. The increased collagen synthesis induced by AngⅡ(10-7mol) was inhibited by Losartan((10-10mol~10-6mol) i n a concentration dependent manner. Conclusion Proliferation of CFb were not influenced by AngⅡ and Bena. Collagen synthesis of CFb was promoted by AngⅡ and inhibited by Bena. Collagen synthesis of CFb in duced by AngⅡ was inhibited by losartan.%目的 观察血管紧张素Ⅱ(AngⅡ)、苯那普利(Benazepril,Bena)、洛沙坦(Losartan)对培养的SHR和WKY大鼠心肌成纤维细胞(CFb)增殖及合成胶原的影响。 方法 采用组织块贴壁法培养CFb,分别测定AngⅡ、Bena干预下CFb的增殖情况, AngⅡ、Bena和Losartan干预下CFb的3H-脯氨酸(3H-Proline)掺入量。 结果 各种浓度的AngⅡ(10-10mol~10-6mol)和Bena(10-9mol~10-5m ol)对CFb的细胞数显著增殖无影响, AngⅡ呈浓度依赖性促进CFb的3H-Proline掺入量,Bena抑制CFb的3H-Proline掺入量,Losartan呈浓度依赖性抑制10-7mol AngⅡ诱导的CFb的胶原合成。 结论 AngⅡ和Bena不影响CFb 的增殖,AngⅡ对CFb的胶原合成有促进作用,Bena抑制CFb 胶原合成,Losartan可拮抗AngⅡ的作用。

  11. 基质细胞衍生因子1α培养心肌细胞和成纤维细胞的增殖与迁移*%Proliferation and migration of cardiomyocytes and cardiac fibroblasts cultured with stromal cell-derived factor-1 alpha

    Institute of Scientific and Technical Information of China (English)

    侯传举; 齐岩梅; 张端珍; 王琦光; 崔春生; 匡丽; 陈兵

    2013-01-01

    BACKGROUND: New wound in the border of defected hearts can promote self-healing, and stromal cel -derived factor-1 alpha can promote angiogenesis and cardiac function. OBJECTIVE: To investigate the effects of mechanical injury and stromal cel -derived factor-1 alpha on the proliferation of cardiomyocytes and the effects of cardiomyocytes on chemotactic migration of cardiac fibroblasts. METHODS: Rat cardiomyocytes and cardiac fibroblasts were cultured primarily. Rat cardiomyocytes were injured mechanical y by scratching, and treated with 10-160 μg/L stromal cel -derived factor-1 alpha. The Cel Counting Kit-8 assay was employed to evaluate the proliferation of cardiomyocytes and the Transwel migration assay was used to detect the chemotaxis and migration capability of cardiac fibroblasts. RESULTS AND CONCLUSION: Under mechanical injury condition, stromal cel -derived factor-1 alpha significantly promoted the growth of cardiomyocytes with the maximum effect occurring at 80 μg/L. In addition, mechanical y injured and stromal cel -derived factor-1 alpha cultured cardiomyocytes remarkably promoted the migration of cardiac fibroblasts in a time-dependent fashion. These findings indicate that stromal cel -derived factor-1 alpha could promote the growth of cardiomyocytes under mechanical injury condition; otherwise, mechanical y injured and stromal cel -derived factor-1 alpha cultured cardiomyocytes could promote the migration of cardiac fibroblasts.%  背景:在心脏缺损边缘造成新创面能够促进缺损自愈,基质细胞衍生因子1α能够促进血管生成以及心脏功能。目的:探讨物理损伤及基质细胞衍生因子1α对心肌细胞增殖的影响及心肌细胞对心脏成纤维细胞迁移的趋化作用。方法:原代分离培养大鼠心肌细胞和心脏成纤维细胞,采用刮伤法建立物理损伤模型,并应用10-160μg/L 的基质细胞衍生因子1α进行干预;CCK-8法检测心

  12. Matrix Metalloproteinase 9 Secreted by Hypoxia Cardiac Fibroblasts Triggers Cardiac Stem Cell Migration In Vitro

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    Qing Gao

    2015-01-01

    Full Text Available Cessation of blood supply due to myocardial infarction (MI leads to complicated pathological alteration in the affected regions. Cardiac stem cells (CSCs migration plays a major role in promoting recovery of cardiac function and protecting cardiomyocytes in post-MI remodeling. Despite being the most abundant cell type in the mammalian heart, cardiac fibroblasts (CFs were underestimated in the mechanism of CSCs migration. Our objective in this study is therefore to investigate the migration related factors secreted by hypoxia CFs in vitro and the degree that they contribute to CSCs migration. We found that supernatant from hypoxia induced CFs could accelerate CSCs migration. Four migration-related cytokines were reported upregulated both in mRNA and protein levels. Upon adding antagonists of these cytokines, the number of migration cells significantly declined. When the cocktail antagonists of all above four cytokines were added, the migration cells number reduced to the minimum level. Besides, MMP-9 had an important effect on triggering CSCs migration. As shown in our results, MMP-9 induced CSCs migration and the underlying mechanism might involve TNF-α signaling which induced VEGF and MMP-9 expression.

  13. Cardiac Fibroblasts Aggravate Viral Myocarditis: Cell Specific Coxsackievirus B3 Replication

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    Diana Lindner

    2014-01-01

    Full Text Available Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3 and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-β was significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase compared to cardiomyocytes (14-fold increase between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.

  14. Protective effect of kaempferol on LPS plus ATP-induced inflammatory response in cardiac fibroblasts.

    Science.gov (United States)

    Tang, Xi-Lan; Liu, Jian-Xun; Dong, Wei; Li, Peng; Li, Lei; Hou, Jin-Cai; Zheng, Yong-Qiu; Lin, Cheng-Ren; Ren, Jun-Guo

    2015-02-01

    Inflammatory response is an important mechanism in the pathogenesis of cardiovascular diseases. Cardiac fibroblasts play a crucial role in cardiac inflammation and might become a potential therapeutic target in cardiovascular diseases. Kaempferol, a flavonoid commonly existing in many edible fruits, vegetables, and Chinese herbs, is well known to possess anti-inflammatory property and thus has a therapeutic potential for the treatment of inflammatory diseases. To date, the effect of kaempferol on cardiac fibroblasts inflammation is unknown. In this study, we investigated the anti-inflammatory effect of kaempferol on lipopolysaccharide (LPS) plus ATP-induced cardiac fibroblasts and explored the underlying mechanisms. Our results showed that kaempferol at concentrations of 12.5 and 25 μg/mL significantly suppressed the release of TNF-α, IL-1β, IL-6, and IL-18 and inhibited activation of NF-κB and Akt in LPS plus ATP-induced cardiac fibroblasts. These findings suggest that kaempferol attenuates cardiac fibroblast inflammation through suppression of activation of NF-κB and Akt.

  15. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

    Science.gov (United States)

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac

    2016-01-01

    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  16. Inscribing Optical Excitability to Non-Excitable Cardiac Cells: Viral Delivery of Optogenetic Tools in Primary Cardiac Fibroblasts

    Science.gov (United States)

    Yu, Jinzhu; Entcheva, Emilia

    2016-01-01

    We describe in detail a method to introduce optogenetic actuation tools, a mutant version of channelrhodopsin- 2, ChR2(H134R), and archaerhodopsin (ArchT), into primary cardiac fibroblasts (cFB) in vitro by adenoviral infection to yield quick, robust, and consistent expression. Instructions on adjusting infection parameters such as the multiplicity of infection and virus incubation duration are provided to generalize the method for different lab settings or cell types. Specific conditions are discussed to create hybrid co-cultures of the optogenetically modified cFB and non-transformed cardiomyocytes to obtain light- sensitive excitable cardiac syncytium, including stencil-patterned cell growth. We also describe an all-optical framework for the functional testing of responsiveness of these opsins in cFB. The presented methodology provides cell-specific tools for the mechanistic investigation of the functional bioelectric contribution of different non-excitable cells in the heart and their electrical coupling to cardiomyocytes under different conditions. PMID:26965132

  17. Fibroblast proliferation alters cardiac excitation conduction and contraction: a computational study.

    Science.gov (United States)

    Zhan, He-qing; Xia, Ling; Shou, Guo-fa; Zang, Yun-liang; Liu, Feng; Crozier, Stuart

    2014-03-01

    In this study, the effects of cardiac fibroblast proliferation on cardiac electric excitation conduction and mechanical contraction were investigated using a proposed integrated myocardial-fibroblastic electromechanical model. At the cellular level, models of the human ventricular myocyte and fibroblast were modified to incorporate a model of cardiac mechanical contraction and cooperativity mechanisms. Cellular electromechanical coupling was realized with a calcium buffer. At the tissue level, electrical excitation conduction was coupled to an elastic mechanics model in which the finite difference method (FDM) was used to solve electrical excitation equations, and the finite element method (FEM) was used to solve mechanics equations. The electromechanical properties of the proposed integrated model were investigated in one or two dimensions under normal and ischemic pathological conditions. Fibroblast proliferation slowed wave propagation, induced a conduction block, decreased strains in the fibroblast proliferous tissue, and increased dispersions in depolarization, repolarization, and action potential duration (APD). It also distorted the wave-front, leading to the initiation and maintenance of re-entry, and resulted in a sustained contraction in the proliferous areas. This study demonstrated the important role that fibroblast proliferation plays in modulating cardiac electromechanical behaviour and which should be considered in planning future heart-modeling studies.

  18. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

    Science.gov (United States)

    Zhang, Xi; Shen, Man-Ru; Xu, Zhen-Dong; Hu, Zhe; Chen, Chao; Chi, Ya-Li; Kong, Zhen-Dong; Li, Zi-Fu; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Zhang, Chuan-Sen

    2014-05-01

    Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the

  19. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    Science.gov (United States)

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. 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...... that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5...... 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...

  1. Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts.

    Science.gov (United States)

    Drummond, Christopher A; Hill, Michael C; Shi, Huilin; Fan, Xiaoming; Xie, Jeffrey X; Haller, Steven T; Kennedy, David J; Liu, Jiang; Garrett, Michael R; Xie, Zijian; Cooper, Christopher J; Shapiro, Joseph I; Tian, Jiang

    2016-03-01

    Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

  2. Clinical Experience with Chitosan Matrix and Cultured Fibroblasts for Burns

    Directory of Open Access Journals (Sweden)

    Gaziza Danlybayeva

    2014-12-01

    Full Text Available Introduction. Burns are an important public health challenge due to the frequency of getting burns in day-to-day life, occupational hazards, and catastrophes. Treatment of burns is complex and is associated with high morbidity and mortality. Duration and complexity of burn treatment require finding new ways of curing and rehabilitating burns. The result of burn treatment plays a significant role in post-traumatic status of a patient and his or her consequent adaptation in society. Chitosan is a natural safe non-toxic product compatible with human tissues, characterized by hydrosorbid, anticoagulant, antibacterial, and wound healing features. The study aims to  show a clinical application of chitosan-pectin scaffold with cultured human skin fibroblasts in the treatment of deep burns.Methods. The substrate was prepared by dissolving 3% chitosan in 0.5N acetic acid, which was then mixed with 3% solution of pectin dissolved in distillated water. Chitosan film was formed in a Petri dish for 20-24 hours at 20-25 °C. After drying the film, cultured allogeneic fibroblasts (patent number RK-25091 were seeded on its surface.Results. The results from an in vitro culture study showed that human allogeneic fibroblasts could adhere well and grow on the selected scaffold with a typical morphology. During autodermoplasty surgery, cultured allogeneic fibroblasts were applied on granulating wounds of 9 patients with IIIA to IVB degree burns and limited donor resources. Wounds treated with the fibroblast-seeded scaffold among all patients provided the highest level of re-epithelialization (day 5, in comparison to cell-free scaffold (day 7 and untreated surface of wounds (day 10.Conclusion. Our results indicate the potential use of chitosan for wound healing due to its allogenic fibroblast adherence to scaffolding as well as high epithelization. This warrants further studies on chitosan for use in wounds resulting from third and fourth degree burns.

  3. Preclinical safety studies on autologous cultured human skin fibroblast transplantation.

    Science.gov (United States)

    Zeng, Wei; Zhang, Shuying; Liu, Dai; Chai, Mi; Wang, Jiaqi; Zhao, Yuming

    2014-01-01

    Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts.

  4. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

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    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  5. Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

    Science.gov (United States)

    Zhang, Yan; Kanter, Evelyn M; Laing, James G; Aprhys, Colette; Johns, David C; Kardami, Elissavet; Yamada, Kathryn A

    2008-09-01

    Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

  6. Fibroblast growth factor 21 as a possible endogenous factor inhibits apoptosis in cardiac endothelial cells

    Institute of Scientific and Technical Information of China (English)

    L(U) Yun; ZHANG Ying-chuan; LIU Jing-hua; ZHANG Li-ke; DU Jie; ZENG Xiang-jun; HAO Gang; HUANG Ji; ZHAO Dong-hui; WANG Guo-zhong

    2010-01-01

    Background Fibroblast growth factor 21 (FGF21) is a new member of FGF super family that is an important endogenous regulator for systemic glucose and lipid metabolism. This study aimed to explore whether FGF21 reduces atherosclerotic injury and prevents endothelial dysfunction as an independent protection factor.Methods The present study was designed to investigate the changes of FGF21 levels induced by oxidized-low density lipoprotein (ox-LDL), and the changes of apoptosis affected by regulating FGF21 expression. The FGF21 mRNA levels of cultured cardiac microvascular endothelial cells (CMECs) were determined by real time-PCR and the protein concentration in culture media was detected by enzyme-linked immunosorbent assay. We analyzed the different expression levels of untreated controls and CMFCs incubated with ox-LDL, and the changes of CMECs apoptosis initiated by the enhancement or suppression of FGF21 levels.Results The secretion levels of FGF21 mRNA and protein were significantly upregulated in CMECs incubated with ox-LDL. Furthermore, FGF21 levels increased by 200 μmol/L bezafibrate could reduce CMECs apoptosis, and inhibit FGF21 expression by shRNA induced apoptosis (P <0.05).Conclusions FGF21 may be a signal of injured target tissue, and may play physiological roles in improving the endothelial function at an early stage of atherosclerosis and in stopping the development of coronary heart disease.

  7. Stimulation of reactive oxygen species and collagen synthesis by angiotensin II in cardiac fibroblasts.

    Science.gov (United States)

    Lijnen, Paul J; van Pelt, Jos F; Fagard, Robert H

    2012-02-01

    Superoxide anion generated by NAD(P)H-oxidase has an important role in the pathogenesis of cardiovascular diseases and scavenging superoxide anion can be considered as a reasonable therapeutic strategy. In hypertensive heart diseases there is a mutual reinforcement of reactive oxygen species (ROS) and angiotensin II (ANG II). ANG II increases the NAD(P)H-dependent superoxide anion production and the intracellular generation of ROS in cardiac fibroblasts and apocynin, a membrane NAD(P)H oxidase inhibitor, abrogates this rise. ANG II also stimulates the collagen production, the collagen I and III content and mRNA expression in cardiac fibroblasts and apocynin abolishes this induction. In this review we demonstrate that scavenging superoxide anion by tempol or EUK-8 or administration of PEG-superoxide dismutase (SOD) inhibits collagen production in cardiac fibroblasts. On the contrary increasing superoxide anion formation by inhibition of SOD stimulates collagen production. A vital role of SOD and the generated ROS can be suggested in the regulation and organization of collagen in cardiac fibroblasts. Specific pharmacological intervention with SOD mimetics can probably be an alternative approach for reducing myocardial fibrosis.

  8. Fibroblast growth factor homologous factors in the heart: a potential locus for cardiac arrhythmias.

    Science.gov (United States)

    Wei, Eric Q; Barnett, Adam S; Pitt, Geoffrey S; Hennessey, Jessica A

    2011-10-01

    The four fibroblast growth factor homologous factors (FHFs; FGF11-FGF14) are intracellular proteins that bind and modulate voltage-gated sodium channels (VGSCs). Although FHFs have been well studied in neurons and implicated in neurologic disease, their role in cardiomyocytes was unclear until recently. This review discusses the expression profile and function of FHFs in mouse and rat ventricular cardiomyocytes. Recent data show that FGF13 is the predominant FHF in the murine heart, directly binds the cardiac VGSC α subunit, and is essential for normal cardiac conduction. FHF loss-of-function mutations may be unrecognized causes of cardiac arrhythmias, such as long QT and Brugada syndromes.

  9. Resveratrol inhibits high glucose induced collagen upregulation in cardiac fibroblasts through regulating TGF-β1-Smad3 signaling pathway.

    Science.gov (United States)

    Liu, Junhui; Zhuo, Xiaozhen; Liu, Weimin; Wan, Zhaofei; Liang, Xiao; Gao, Shanshan; Yuan, Zuyi; Wu, Yue

    2015-02-05

    Cardiac fibrosis is a common pathological process presented in a variety of diseases, including hypertension and diabetes. Cardiac fibroblasts (CFs) have been identified as the most important participants in the development of cardiac fibrosis. Exposure of cultured CFs to high glucose (HG) or angiotensin II (Ang II) resulted in increased collagen synthesis. Resveratrol (Res) is a natural polyphenol exhibiting anti-fibrosis effects in a number of different organs fibrosis process, whether Res can prevent HG and Ang II induced fibrosis response in CFs remains unclear. The aim of this work was to evaluate the effects of Res in HG and Ang II induced fibrosis response in CFs. We cultured rat CFs in either normal glucose (5.6 mM) or HG (25 mM) media in the presence of Res or not and the changes in collagens synthesis and TGF-β1 production were assessed by Real-time PCR, Western blotting, and enzyme linked immunosorbent assay (ELISA). Furthermore, normal and diabetic mice (induced by single dose of streptozotocin (100 mg/kg) via tail vein) receiving Res (10 mg/kg) were used to explore the effects of Res on cardiac fibrosis in vivo. Masson staining and immunohistochemistry were performed to visualize cardiac collagen deposition. Results indicate that CFs exposed to HG condition shows enhanced proliferation rate. Furthermore, in the presence of HG or Ang II, CFs exhibited increased collagens synthesis and TGF-β1 production. And these effects were abolished by Res intervention. In vivo results show that diabetic mice exhibit increased collagen deposition in the cardiac compared with the normal mice. And this change was prevented by the treatment of Res. These results suggest that Res possesses a potential antifibrogenic effect in hypertension and diabetes-related cardiac fibrosis. Moreover, the action mechanism is probably associated with its ability to reduce TGF-β1 content in CFs.

  10. Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

    Directory of Open Access Journals (Sweden)

    Giacomo Palazzolo

    2016-01-01

    Full Text Available The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs. We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C, known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

  11. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  12. Temporal alterations in cardiac fibroblast function following induction of pressure overload

    Science.gov (United States)

    Stewart, James A.; Massey, Erin P.; Fix, Charity; Zhu, Jinyu; Goldsmith, Edie C.

    2014-01-01

    Increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis. While numerous studies have focused on the mechanisms of myocyte hypertrophy, comparatively little is known regarding the response of the interstitial fibroblasts to increased cardiovascular load. Fibroblasts are the most numerous cell type in the mammalian myocardium and have long been recognized as producing the majority of the myocardial extracellular matrix. It is only now becoming appreciated that other aspects of fibroblast behavior are important to overall cardiac function. The present studies were performed to examine the temporal alterations in fibroblast activity in response to increased cardiovascular load. Rat myocardial fibroblasts were isolated at specific time-points (3, 7, 14, and 28 days) after induction of pressure overload by abdominal aortic constriction. Bioassays were performed to measure specific parameters of fibroblast function including remodeling and contraction of 3-dimensional collagen gels, migration, and proliferation. In addition, the expression of extracellular matrix receptors of the integrin family was examined. Myocardial hypertrophy and fibrosis were evident within 7 days after constriction of the abdominal aorta. Collagen gel contraction, migration, and proliferation were enhanced in fibroblasts from pressure-overloaded animals compared to fibroblasts from sham animals. Differences in fibroblast function and protein expression were evident within 7 days of aortic constriction, concurrent with the onset of hypertrophy and fibrosis of the intact myocardium. These data provide further support for the idea that rapid and dynamic changes in fibroblast phenotype accompany and contribute to the progression of cardiovascular disease. PMID:20217135

  13. Characterization of epicardial-derived cardiac interstitial cells: differentiation and mobilization of heart fibroblast progenitors.

    Directory of Open Access Journals (Sweden)

    Adrián Ruiz-Villalba

    Full Text Available The non-muscular cells that populate the space found between cardiomyocyte fibers are known as 'cardiac interstitial cells' (CICs. CICs are heterogeneous in nature and include different cardiac progenitor/stem cells, cardiac fibroblasts and other cell types. Upon heart damage CICs soon respond by initiating a reparative response that transforms with time into extensive fibrosis and heart failure. Despite the biomedical relevance of CICs, controversy remains on the ontogenetic relationship existing between the different cell kinds homing at the cardiac interstitium, as well as on the molecular signals that regulate their differentiation, maturation, mutual interaction and role in adult cardiac homeostasis and disease. Our work focuses on the analysis of epicardial-derived cells, the first cell type that colonizes the cardiac interstitium. We present here a characterization and an experimental analysis of the differentiation potential and mobilization properties of a new cell line derived from mouse embryonic epicardium (EPIC. Our results indicate that these cells express some markers associated with cardiovascular stemness and retain part of the multipotent properties of embryonic epicardial derivatives, spontaneously differentiating into smooth muscle, and fibroblast/myofibroblast-like cells. Epicardium-derived cells are also shown to initiate a characteristic response to different growth factors, to display a characteristic proteolytic expression profile and to degrade biological matrices in 3D in vitro assays. Taken together, these data indicate that EPICs are relevant to the analysis of epicardial-derived CICs, and are a god model for the research on cardiac fibroblasts and the role these cells play in ventricular remodeling in both ischemic or non/ischemic myocardial disease.

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

    Directory of Open Access Journals (Sweden)

    Xiaodong Pan

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

  15. 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)

  16. IL-1β suppresses TGF-β-mediated myofibroblast differentiation in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Brønnum, Hasse; Eskildsen, Tilde; Andersen, Ditte Caroline;

    2013-01-01

    Cardiac fibrosis is a maladaptive response of the injured myocardium and is mediated through a complex interplay between molecular triggers and cellular responses. Interleukin (IL)-1β is a key inflammatory inducer in cardiac disease and promotes cell invasion and cardiomyocyte injury, but little...... a differentiation model of ventricular fibroblasts, we found that IL-1β instigated substantial anti-fibrogenic effects. In specific, IL-1β reduced proliferation, matrix activity, cell motility and α-smooth muscle actin expression, which are all hallmarks of myoFb differentiation. These findings suggest that IL-1β...

  17. Vitronectin-binding PAI-1 protects against the development of cardiac fibrosis through interaction with fibroblasts.

    Science.gov (United States)

    Zhong, Jianyong; Yang, Hai-Chun; Kon, Valentina; Fogo, Agnes B; Lawrence, Daniel A; Ma, Ji

    2014-06-01

    Plasminogen activator inhibitor-1 (PAI-1) promotes or abates fibrotic processes occurring in different organs. Binding of PAI-1 to vitronectin, an extracellular matrix component, may inhibit vitronectin-integrin complex-mediated cellular responses in pathophysiological conditions. To investigate the importance of plasmin suppression vs vitronectin-binding pathways of PAI-1 in cardiac fibrosis, we studied uninephrectomized mice fed a high salt diet and infused with angiotensin II (Ang II) together with different PAI-1 variants, including PAI-1AK (AK) that inhibits plasminogen activators but does not bind vitronectin, PAI-1RR (RR) that binds vitronectin but does not have protease inhibitory effects or control PAI-1 (CPAI), the control mutant that has similar molecular backbone and half-life as AK and RR while retaining all functions of native PAI-1. Compared with RR and CPAI, non-vitronectin-binding AK significantly increased expression of cardiac fibroblast marker, periostin (Ang+AK 8.40±3.55 vs Ang+RR 2.23±0.44 and Ang+CPAI 2.33±0.12% positive area, both PPAI-1 against fibrosis, fibroblasts from normal adult human ventricles were stimulated with Ang and different PAI-1 variants. Protease inhibitory AK and CPAI increased supernatant fibronectin, while decreasing plasminogen activator/plasmin activities and matrix metalloproteinase. RR and CPAI variants significantly reduced fibroblast expression of integrin β3, vitronectin level in the supernatant and fibroblast adhesion to vitronectin compared with the non-vitronectin-binding AK. Further, RR and CPAI preserved apoptotic, decreased anti-apoptotic and proliferative activities in fibroblasts. Thus, PAI-1 promotes or protects against development of cardiac fibrosis differentially through the protease inhibitory pathway or through its binding to vitronectin.

  18. Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Catalán, Mabel; Smolic, Christian [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Contreras, Ariel [Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Ayala, Pedro; Olmedo, Ivonne; Copaja, Miguel; Boza, Pía; Vivar, Raúl; Avalos, Yennifer [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Lavandero, Sergio [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX (United States); Velarde, Victoria [Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago (Chile); Díaz-Araya, Guillermo, E-mail: gadiaz@ciq.uchile.cl [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile)

    2012-06-15

    Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. Methods: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-β1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca{sup +2} levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. Results: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca{sup 2+} levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca{sup 2+} levels in CF; however, after preincubation for 1 h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca{sup 2+} levels. Finally, DAKD increased intracellular Ca{sup 2+} levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. Conclusion: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was

  19. Telmisartan inhibited angiotensin II-induced collagen metabolic imbalance without directly targeting TGF-β 1/Smad signaling pathway in cardiac fibroblasts.

    Science.gov (United States)

    Zhang, Y; Zhao, N A; Wang, J K; Zhu, S M; Zhu, H L; Liu, B; Cui, Q W; Guan, G C; Tian, G

    2015-12-01

    Cardiac fibrosis is an important pathological process of cardiac remodeling. A large number of studies have shown that telmisartan can attenuate cardiac fibrosis through acting on angiotensin II 1 receptor (AT1R), and TGF-β 1/Smad signaling molecule is an important pathway to achieve this effect. The aim of the study was to clarify whether, with excessive activation of RAAS system, telmisartan could also directly target TGF-β 1/Smad signaling pathway to have the function of anti-cardiac fibrosis. In this study, neonatal rat cardiac fibroblasts were cultured and AngII or TGF-β 1 was administered for treatment or pre-incubation, and then telmisartan was used for 24 hours' incubation. Western blot and enzyme-linked immunosorbent assay (ELISA) tests were performed to detect protein expressions. The results showed that telmisartan could inhibit collagen synthesis and collagen metabolic imbalance under the effect of Ang II, but telmisartan could not have such function in TGF-β 1-induced cardiac fibroblasts. It was further confirmed by western blot method that telmisartan could inhibit TGF-β 1/Smad signaling molecule expression under the effect of Ang II, but telmisartan had no effect on TGF-β 1-induced Smad signaling molecule expression. According to the present study telmisartan played a role of anticardiac fibrosis without directly targeting TGF-β 1/Smad signaling pathway molecule.

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

    Directory of Open Access Journals (Sweden)

    Emond Mary

    2007-09-01

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

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

    Science.gov (United States)

    Yao, Zizhen; Jaeger, Jochen C; Ruzzo, Walter L; Morale, Cecile Z; Emond, Mary; Francke, Uta; Milewicz, Dianna M; Schwartz, Stephen M; Mulvihill, Eileen R

    2007-01-01

    Background Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater. PMID:17850668

  2. Functional 3-D cardiac co-culture model using bioactive chitosan nanofiber scaffolds.

    Science.gov (United States)

    Hussain, Ali; Collins, George; Yip, Derek; Cho, Cheul H

    2013-02-01

    , the cardiomyocyte-fibroblast co-cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long-term culture. The Cx43 expression in the fibroblast co-culture was higher than the cardiomyocytes mono-culture and endothelial cells co-culture. In addition, fibroblast co-cultures demonstrated synchronized contractions involving large tissue-like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3-D cardiac co-culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3-D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications.

  3. Effects of interleukin-1 on cardiac fibroblast function: relevance to post-myocardial infarction remodelling.

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    Turner, Neil A

    2014-01-01

    The cardiac fibroblast (CF) is a multifunctional and heterogeneous cell type that plays an essential role in regulating cardiac development, structure and function. Following myocardial infarction (MI), the myocardium undergoes complex structural remodelling in an attempt to repair the damaged tissue and overcome the loss of function induced by ischemia/reperfusion injury. Evidence is emerging that CF play critical roles in all stages of post-MI remodelling, including the initial inflammatory phase that is triggered in response to myocardial damage. CF are particularly responsive to the proinflammatory cytokine interleukin-1 (IL-1) whose levels are rapidly induced in the myocardium after MI. Studies from our laboratory in recent years have sought to evaluate the functional effects of IL-1 on human CF function and to determine the underlying molecular mechanisms. This review summarises these data and sets it in the context of post-MI cardiac remodelling, identifying the fibroblast as a potential therapeutic target for reducing adverse cardiac remodelling and its devastating consequences. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Functional Alterations of Ion Channels From Cardiac Fibroblasts in Heart Diseases

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    Gracious R. Ross

    2016-11-01

    Full Text Available In an aged population, cardiovascular disease is the leading cause of fatality and morbidity. Age-related fibrotic remodeling of the heart contributes to progressive myocardial dysfunction. Cardiac fibroblasts (CF, responsible for the maintenance of extracellular matrix and fibrosis process, play an important role in cardiac health and disease. CFs influence myocardial function by their chemical, electrical and mechanical interactions with cardiomyocytes through extracellular matrix deposition or secretion of cytokines and growth factors. These, in turn, are modulated by ion channels, macromolecular pores in the plasma membrane that allow selective ionic fluxes of major ions like K+, Ca2+, Na+ or Cl-, which affect membrane potential and cellular signal transduction. The importance of ion channels in modulating various functions of CFs, including proliferation, differentiation, secretion and apoptosis, is being recognized from recent studies of CFs from animal models and tissue from patients with various cardiac pathologies. Understanding the role of ion channels in CFs under physiological conditions and their alterations in age-related cardiac diseases may help facilitate development of novel therapeutic strategies to limit cardiac fibrosis and its adverse effect on myocardial function. This narrative review summarizes the knowledge gained thus far on ion channels in CFs and their relationship with cardiac diseases in human and experimental animal models.

  5. Biologic characteristics of fibroblast cells cultured from the knee ligaments

    Institute of Scientific and Technical Information of China (English)

    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿

    2002-01-01

    Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

  6. β3 integrin in cardiac fibroblast is critical for extracellular matrix accumulation during pressure overload hypertrophy in mouse.

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    Sundaravadivel Balasubramanian

    Full Text Available The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT and β3 integrin null (β3-/- mice for in vivo pressure overload (PO and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1 expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis stimulation. Our results showed that β3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

  7. Relaxin Prevents Cardiac Fibroblast-Myofibroblast Transition via Notch-1-Mediated Inhibition of TGF-β/Smad3 Signaling

    Science.gov (United States)

    Sassoli, Chiara; Chellini, Flaminia; Pini, Alessandro; Tani, Alessia; Nistri, Silvia; Nosi, Daniele; Zecchi-Orlandini, Sandra; Bani, Daniele; Formigli, Lucia

    2013-01-01

    The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion

  8. Relaxin prevents cardiac fibroblast-myofibroblast transition via notch-1-mediated inhibition of TGF-β/Smad3 signaling.

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

    Full Text Available The hormone relaxin (RLX is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT, a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this

  9. The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts.

    Science.gov (United States)

    Schultz, Francisca; Hasan, Alveera; Alvarez-Laviada, Anita; Miragoli, Michele; Bhogal, Navneet; Wells, Sarah; Poulet, Claire; Chambers, Jenny; Williamson, Catherine; Gorelik, Julia

    2016-01-01

    Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts. Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential. We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells. UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts

  10. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  11. Effects of direct Renin inhibition on myocardial fibrosis and cardiac fibroblast function.

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    Hui Zhi

    Full Text Available Myocardial fibrosis, a major pathophysiologic substrate of heart failure with preserved ejection fraction (HFPEF, is modulated by multiple pathways including the renin-angiotensin system. Direct renin inhibition is a promising anti-fibrotic therapy since it attenuates the pro-fibrotic effects of renin in addition to that of other effectors of the renin-angiotensin cascade. Here we show that the oral renin inhibitor aliskiren has direct effects on collagen metabolism in cardiac fibroblasts and prevented myocardial collagen deposition in a non-hypertrophic mouse model of myocardial fibrosis. Adult mice were fed hyperhomocysteinemia-inducing diet to induce myocardial fibrosis and treated concomitantly with either vehicle or aliskiren for 12 weeks. Blood pressure and plasma angiotensin II levels were normal in control and hyperhomocysteinemic mice and reduced to levels lower than observed in the control group in the groups treated with aliskiren. Homocysteine-induced myocardial matrix gene expression and fibrosis were also prevented by aliskiren. In vitro studies using adult rat cardiac fibroblasts also showed that aliskiren attenuated the pro-fibrotic pattern of matrix gene and protein expression induced by D,L, homocysteine. Both in vivo and in vitro studies demonstrated that the Akt pathway was activated by homocysteine, and that treatment with aliskiren attenuated Akt activation. In conclusion, aliskiren as mono-therapy has potent and direct effects on myocardial matrix turnover and beneficial effects on diastolic function.

  12. The cardiac fibroblast: Origin, identity and role in homeostasis and disease.

    Science.gov (United States)

    Furtado, Milena B; Costa, Mauro W; Rosenthal, Nadia A

    2016-09-01

    The mammalian heart is responsible for supplying blood to two separate circulation circuits in a parallel manner. This design provides efficient oxygenation and nutrients to the whole body through the left-sided pump, while the right-sided pump delivers blood to the pulmonary circulation for re-oxygenation. In order to achieve this demanding job, the mammalian heart evolved into a highly specialised organ comprised of working contractile cells or cardiomyocytes, a directional and insulated conduction system, capable of independently generating and conducting electric impulses that synchronises chamber contraction, valves that allow the generation of high pressure and directional blood flow into the circulation, coronary circulation, that supplies oxygenated blood for the heart muscle high metabolically active pumping role and inlet/outlet routes, as the venae cavae and pulmonary veins, aorta and pulmonary trunk. This organization highlights the complexity and compartmentalization of the heart. This review will focus on the cardiac fibroblast, a cell type until recently ignored, but that profoundly influences heart function in its various compartments. We will discuss current advances on definitions, molecular markers and function of cardiac fibroblasts in heart homeostasis and disease.

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

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    Restu Syamsul Hadi

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2014-08-01

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

  15. Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Tamaki, Yodo; Iwanaga, Yoshitaka; Niizuma, Shinichiro

    2013-01-01

    Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relati...... interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis....... relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown...

  16. On-chip constructive cell-Network study (I: Contribution of cardiac fibroblasts to cardiomyocyte beating synchronization and community effect

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    Yasuda Kenji

    2011-05-01

    Full Text Available Abstract Backgrounds To clarify the role of cardiac fibroblasts in beating synchronization, we have made simple lined-up cardiomyocyte-fibroblast network model in an on-chip single-cell-based cultivation system. Results The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1 propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2 fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3 the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time. Conclusions The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 'community effect', which enhances synchronization and stability of their beating rhythms.

  17. Substrate stiffness-regulated matrix metalloproteinase output in myocardial cells and cardiac fibroblasts: implications for myocardial fibrosis.

    Science.gov (United States)

    Xie, Jing; Zhang, Quanyou; Zhu, Ting; Zhang, Yanyan; Liu, Bailin; Xu, Jianwen; Zhao, Hucheng

    2014-06-01

    Cardiac fibrosis, an important pathological feature of structural remodeling, contributes to ventricular stiffness, diastolic dysfunction, arrhythmia and may even lead to sudden death. Matrix stiffness, one of the many mechanical factors acting on cells, is increasingly appreciated as an important mediator of myocardial cell behavior. Polydimethylsiloxane (PDMS) substrates were fabricated with different stiffnesses to mimic physiological and pathological heart tissues, and the way in which the elastic modulus of the substrate regulated matrix-degrading gelatinases in myocardial cells and cardiac fibroblasts was explored. Initially, an increase in cell spreading area was observed, concomitant with the increase in PDMS stiffness in both cells. Later, it was demonstrated that the MMP-2 gene expression and protein activity in myocardial cells and cardiac fibroblasts can be enhanced with an increase in PDMS substrate stiffness and, moreover, such gene- and protein-related increases had a significant linear correlation with the elastic modulus. In comparison, the MMP-9 gene and protein expressions were up-regulated in cardiac fibroblasts only, not in myocardial cells. These results implied that myocardial cells and cardiac fibroblasts in the myocardium could sense the stiffness in pathological fibrosis and showed a differential but positive response in the expression of matrix-degrading gelatinases when exposed to an increased stiffening of the matrix in the microenvironment. The phenomenon of cells sensing pathological matrix stiffness can help to increase understanding of the mechanism underlying myocardial fibrosis and may ultimately lead to planning cure strategies.

  18. Trypanosoma cruzi highjacks TrkC to enter cardiomyocytes and cardiac fibroblasts while exploiting TrkA for cardioprotection against oxidative stress.

    Science.gov (United States)

    Aridgides, Daniel; Salvador, Ryan; PereiraPerrin, Mercio

    2013-08-01

    Chronic Chagas cardiomyopathy (CCC), caused by the obligate intracellular protozoan parasite Trypanosoma cruzi, is a major cause of morbidity and mortality in Latin America. CCC begins when T. cruzi enters cardiac cells for intracellular multiplication and differentiation, a process that starts with recognition of host-cell entry receptors. However, the nature of these surface molecules and corresponding parasite counter-receptor(s) is poorly understood. Here we show that antibodies against neurotrophin (NT) receptor TrkC, but not against family members TrkA and TrkB, prevent T. cruzi from invading primary cultures of cardiomyocytes and cardiac fibroblasts. Invasion is also selectively blocked by the TrkC ligand NT-3, and by antagonists of Trk autophosphorylation and downstream signalling. Therefore, these results indicate that T. cruzi gets inside cardiomyocytes and cardiac fibroblasts by activating TrkC preferentially over TrkA. Accordingly, short hairpin RNA interference of TrkC (shTrkC), but not TrkA, selectively prevents T. cruzi from entering cardiac cells. Additionally, T. cruzi parasite-derived neurotrophic factor (PDNF)/trans-sialidase, a TrkC-binding protein, but not family member gp85, blocks entry dose-dependently, underscoring the specificity of PDNF as TrkC counter-receptor in cardiac cell invasion. In contrast to invasion, competitive and shRNA inhibition studies demonstrate that T. cruzi-PDNF recognition of TrkA, but not TrkC on primary cardiomyocytes and the cardiomyocyte cell line H9c2 protects the cells against oxidative stress. Thus, this study shows that T. cruzi via PDNF favours neurotrophin receptor TrkC for cardiac cell entry and TrkA for cardiomyocyte protection against oxidative stress, and suggests a new therapeutic opportunity in PDNF and/or fragments thereof for CCC therapy as entry inhibitors and/or cardioprotection agonists. © 2013 John Wiley & Sons Ltd.

  19. Beryllium toxicity testing in the suspension culture of mouse fibroblasts.

    Science.gov (United States)

    Rössner, P; Bencko, V

    1980-01-01

    Suspension culture of mouse fibroblast cell line L-A 115 was used to test beryllium toxicity in the presence of magnesium ions. Beryllium added to the MEM cultivation medium was bound in a complex with sulphosalicylic acid BeSSA complex, because the use of beryllium chloride turned out to yield ineffective beryllium phosphate that formed macroscopically detectable insoluble opacities. The BeSSA complex was used in the concentration range: 10(-3)--10(-9)M, magnesium was used in 3 concentrations: 10(-1)M, 5 x 10(-2)M and 10(-2)M. Growth curve analysis revealed pronounced beryllium toxicity at the concentration of 10(-3)M, magnesium-produced toxic changes were observed only at the concentration of 10(-1)M. No competition between the beryllium and magnesium ions was recorded. It is assumed that the possible beryllium-magnesium competition was significantly modified by the use of BeSSA complex-bound beryllium.

  20. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    Science.gov (United States)

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  1. Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

    Directory of Open Access Journals (Sweden)

    Zhenxiang Wang

    Full Text Available Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th to 15(th day, keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF, IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

  2. Fibroblast Growth Factor-23 in Obese, Normotensive Adolescents is Associated with Adverse Cardiac Structure

    Science.gov (United States)

    Ali, Farah N.; Falkner, Bonita; Gidding, Samuel S.; Price, Heather E.; Keith, Scott W.; Langman, Craig B.

    2014-01-01

    Objectives Fibroblast growth factor-23 (FGF23) is a biomarker for cardiovascular (CV) disease. Obesity may promote FGF23 production in the absence of chronic kidney disease (CKD). We sought to determine among normotensive African American adolescents, whether FGF23 levels are higher in obese compared with normal weight African American adolescents; and to determine the relationship of FGF23 with markers of cardiac structure and insulin resistance. Study design Cross-sectional data were obtained from a cohort of 130 normotensive, African American adolescents aged 13-18 years old without CKD; 74 were obese; 56 were normal weight. Plasma C-terminal FGF23, fasting glucose and insulin, and hsCRP were measured; participants underwent M-mode echocardiography. Results FGF23 was skewed and approximately normally distributed after natural log transformation (logFGF23). FGF23 levels were higher in obese versus normal weight participants (geometric mean 43 vs. 23 RU/mL, p<0.01). FGF23 values were significantly higher in participants with eccentric or concentric cardiac hypertrophy compared with those without hypertrophy (p<0.01). LogFGF23 directly correlated with BMI, BMI z-score, waist circumference, fasting insulin levels, and HOMA scores. Regression models adjusted for age, sex, and hsCRP suggest that each 10% increase in FGF23 is associated with 1.31 unit increase in LVM (p<0.01), 0.29 unit increase in LVMI (p<0.01), and 0.01 unit increase in left atrial dimension indexed to height (p=0.02). Conclusions In this sample of obese African American adolescents, FGF23 blood levels were associated with abnormal cardiac structure. We postulate that FGF23 may be an early marker of cardiac injury in obese but otherwise healthy African American adolescents. PMID:25063724

  3. Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

    Science.gov (United States)

    Chen, Shao-Rui; Zhang, Wen-Ping; Bao, Jing-Mei; Cheng, Zhong-Bin; Yin, Sheng

    2017-01-01

    Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca(2+), we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

  4. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    Energy Technology Data Exchange (ETDEWEB)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M. (Medical Univ. School of Silesia, Katowice (Poland))

    1990-04-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and (14C)thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil.

  5. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Cecilia Prata

    2017-01-01

    Full Text Available Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  6. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Science.gov (United States)

    Prata, Cecilia; Zambonin, Laura; Rizzo, Benedetta; Vieceli Dalla Sega, Francesco

    2017-01-01

    Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  7. Regenerative material for aneurysm embolization A 3-dimensional culture system of fibroblasts and calcium alginate gel

    Institute of Scientific and Technical Information of China (English)

    Jingdong Zhang; Kan Xu; Jinlu Yu; Jun Wang; Qi Luo

    2011-01-01

    Calcium alginate gel (CAG) has been shown to successfully model aneurysm embolization within a short period of time. However, gradually degrading CAG potentially results in aneurysm recanalization.In the present study, a regenerative embolic material was designed by seeding rat fibroblasts in a CAG. The study investigated the feasibility of constructing a 3-dimensional culture system. The fibroblasts grew well and firmly attached to the CAG. CAG was conducive for fibroblast growth, and resulted in a 3-dimensional culture system. Results show that CAG can be used theoretically as a vascular, regenerative, embolic material.

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

    Directory of Open Access Journals (Sweden)

    Jon M Carthy

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

  9. High molecular weight fibroblast growth factor-2 in the human heart is a potential target for prevention of cardiac remodeling.

    Directory of Open Access Journals (Sweden)

    Jon-Jon Santiago

    Full Text Available Fibroblast growth factor 2 (FGF-2 is a multifunctional protein synthesized as high (Hi- and low (Lo- molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD and 68% (±25 SD of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-AbHi-FGF-2 reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-AbHi-FGF-2 in vitro. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes

  10. Cardiac fibroblasts contribute to myocardial dysfunction in mice with sepsis: the role of NLRP3 inflammasome activation.

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    Wenbo Zhang

    Full Text Available Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3 inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk. We show that treatment of CFs with lipopolysaccharide (LPS induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA] and pharmacological (glyburide inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-challenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.

  11. Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar

    Full Text Available RATIONALE: Thymosin beta-4 (Tβ4 is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. METHODS: Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H(2O(2 in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H(2O(2-induced profibrotic events was evaluated. RESULTS: Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD and catalase and reduction of Bax/Bcl(2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF, collagen type-1 (Col-I and collagen type-3 (Col-III] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2, thereby, preventing H(2O(2-induced profibrotic changes in the myocardium. Further studies are

  12. Inductive role of fibroblastic cell lines in development of the mouse thymus anlage in organ culture.

    Science.gov (United States)

    Itoi, M; Amagai, T

    1998-01-10

    Previously, we have shown that embryonic day 12 thymus anlage cultured alone cannot develop into the mature organ but degenerates. In the present study, we investigated the cause of this insufficient organogenesis of embryonic day 12 thymus anlage in organ culture. We cocultured embryonic day 12 thymus anlages with various cell lines as pellets formed by centrifugation. In coculture with fibroblastic cell lines, but not with thymic epithelial cell lines, embryonic day 12 thymus anlages developed to support full T cell differentiation, and expressed mature stromal cell markers, Ia and Kb. By pellet culture of thymus anlages and fibroblastic cell lines transfected with a beta-galactosidase expression vector, we analyzed the distribution of added fibroblastic cells in pellets. The added fibroblastic cells constituted neither thymic capsule nor septa but disappeared after about 2 weeks in culture. Moreover, immunohistochemical studies indicated that added fibroblastic cells were adjacent to mesenchymal cells of thymus anlage. Our results strongly suggest that added fibroblastic cells support the development of the thymus anlage through interaction with its mesenchymal cells.

  13. Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model.

    Science.gov (United States)

    Majety, Meher; Pradel, Leon P; Gies, Manuela; Ries, Carola H

    2015-01-01

    In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.

  14. MORPHOFUNCTIONAL CHARACTERISTICS OF FIBROBLASTS (MCCOY CELL LINE CULTURED WITH MAGNESIUM PREPARATIONS

    Directory of Open Access Journals (Sweden)

    L. V. Didenko

    2015-01-01

    Full Text Available Aim. To study the effect of magnesium orotate, magnesium/pyridoxine combination and magnesium sulfate on fibroblast morphofunctional characteristics in cell culture of fibroblasts (McCoy line.Material and methods. The study of fibroblasts (McCoy line with the addition of magnesium-containing preparations (magnesium orotate, magnesium/pyridoxine combination, magnesium sulphate to the culture medium was performed using scanning and transmission electron microscopy.Results. When adding into the media magnesium orotate or magnesium/pyridoxine combination, significant changes in morphofunctional state of fibroblasts were noted, which were absent when adding magnesium sulfate. At that fibroblasts significantly promoted synthetic and secretory activity. This was expressed in the formation of amorphous and fibrous components in well-formed cell layers.Conclusion. Stimulation by magnesium ions of the proliferative activity of fibroblasts is shown in the morphological analysis of the effect of magnesium orotate or magnesium in combination with pyridoxine and magnesium sulfate on morphological and functional organization of fibroblasts. Revealed the predominant influence of magnesium orotate, and a less pronounced effect of magnesium in combination with pyridoxine on biosynthetic processes in the cells, including the synthesis of amorphous and fibrous components (protocollagen and elastin of the extracellular matrix founded.

  15. Effect of glutathione on arecanut treated normal human buccal fibroblast culture.

    Directory of Open Access Journals (Sweden)

    Saraswathi T

    2006-01-01

    Full Text Available BACKGROUND: Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential. OBJECTIVE: The present study was undertaken to evaluate the effect of glutathione on arecanut treated human buccal fibroblast culture and its potential as a chemopreventive agent. MATERIALS AND METHODS: Fibroblast culture was done in Dulbecco′s Modified Eagle′s Medium MEM supplemented with 10% Fetal Calf Serum (FCS and antibiotic at 370C degrees in an atmosphere of 5% carbon di-oxide and 95% air. The fibroblast cells were subjected to different concentrations of aqueous extracts of raw and boiled arecanut. Fibroblasts were plated in two 24-well culture plates and in each plate, cells were dividt,ednto 2 groups; 600gg microml of reduced glutathione was added to the first group of cells; subsequently, aqueous extracts of raw and boiled arecanut at least and highest concentrations i.e., 20j. microml and 100lg microml were added to the first group of cells in the respective plates whereas the second group served as a control. The morphological alterations and cell survival were assayed at 24, 48, 72, and 96 hours. Results Morphologically, the initial (10 hours attached fibroblast cells were converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration. The three morphological types of fibroblasts at 48 hours were F-I, F-II and F-III. Aqueous extract of raw arecanut exhibited significant cytotoxicity (p < .0 001 at all time periods studied, when compared against the control values of untreated fibroblasts. Addition of reduced glutathione to cultures showed a significant (p < 0. 001 reduction in cytotoxicity, as indicated by higher optical density values and morphological reversion to the spindle-shaped configuration. CoCONCLUSION:Addition of glutathione reduced the cytotoxic and morphological alterations of the fibroblasts treated with aqueous extracts of both raw and boiled

  16. 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

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired...

  17. 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

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired, in...

  18. 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...

  19. Adenylyl cyclase type 6 overexpression selectively enhances β-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts

    OpenAIRE

    Liu, Xiaoqiu; Thangavel, Muthusamy; Sun, Shu Qiang; Kaminsky, Joseph; Mahautmr, Penden; Stitham, Jeremiah; Hwa, John; Ostrom, Rennolds S.

    2007-01-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor β and inhibited by agents that elevate 3′,5′-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. Th...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...... collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.......Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts...... to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned...

  1. Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta.

    Science.gov (United States)

    Watson, Chris J; Phelan, Dermot; Collier, Patrick; Horgan, Stephen; Glezeva, Nadia; Cooke, Gordon; Xu, Maojia; Ledwidge, Mark; McDonald, Kenneth; Baugh, John A

    2014-06-01

    Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

  2. Cardiac cell culture model as a left ventricle mimic for cardiac tissue generation.

    Science.gov (United States)

    Nguyen, Mai-Dung; Tinney, Joseph P; Yuan, Fangping; Roussel, Thomas J; El-Baz, Ayman; Giridharan, Guruprasad; Keller, Bradley B; Sethu, Palaniappan

    2013-09-17

    A major challenge in cardiac tissue engineering is the delivery of hemodynamic mechanical cues that play a critical role in the early development and maturation of cardiomyocytes. Generation of functional cardiac tissue capable of replacing or augmenting cardiac function therefore requires physiologically relevant environments that can deliver complex mechanical cues for cardiomyocyte functional maturation. The goal of this work is the development and validation of a cardiac cell culture model (CCCM) microenvironment that accurately mimics pressure-volume changes seen in the left ventricle and to use this system to achieve cardiac cell maturation under conditions where mechanical loads such as pressure and stretch are gradually increased from the unloaded state to conditions seen in vivo. The CCCM platform, consisting of a cell culture chamber integrated within a flow loop was created to accomplish culture of 10 day chick embryonic ventricular cardiomyocytes subject to 4 days of stimulation (10 mmHg, ∼13% stretch at a frequency of 2 Hz). Results clearly show that CCCM conditioned cardiomyocytes accelerate cardiomyocyte structural and functional maturation in comparison to static unloaded controls as evidenced by increased proliferation, alignment of actin cytoskeleton, bundle-like sarcomeric α-actinin expression, higher pacing beat rate at lower threshold voltages, and increased shortening. These results confirm the CCCM microenvironment can accelerate immature cardiac cell structural and functional maturation for potential cardiac regenerative applications.

  3. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  4. Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases.

    Science.gov (United States)

    Virtanen, I; Ekblom, P; Laurila, P; Nordling, S; Raivio, K O; Aula, P

    1980-11-01

    The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure. In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for N-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.

  5. High Density Sphere Culture of Adult Cardiac Cells Increases the Levels of Cardiac and Progenitor Markers and Shows Signs of Vasculogenesis

    Directory of Open Access Journals (Sweden)

    Kristina Vukusic

    2013-01-01

    Full Text Available 3D environment and high cell density play an important role in restoring and supporting the phenotypes of cells represented in cardiac tissues. The aim of this study was therefore to investigate the suitability of high density sphere (HDS cultures for studies of cardiomyocyte-, endothelial-, and stem-cell biology. Primary adult cardiac cells from nine human biopsies were cultured using different media for up to 9 weeks. The possibilities to favor a certain cell phenotype and induce production of extra cellular matrix (ECM were studied by histology, immunohistochemistry, and quantitative real-time PCR. Defined media gave significant increase in both cardiac- and progenitor-specific markers and also an intraluminal position of endothelial cells over time. Cardiac media showed indication of differentiation and maturity of HDS considering the ECM production and activities within NOTCH regulation but no additional cardiac differentiation. Endothelial media gave no positive effects on endothelial phenotype but increased proliferation without fibroblast overgrowth. In addition, indications for early vasculogenesis were found. It was also possible to affect the Wnt signaling in HDS by addition of a glycogen synthase kinase 3 (GSK3 inhibitor. In conclusion, these findings show the suitability of HDS as in vitro model for studies of cardiomyocyte-, endothelial-, and stem-cell biology.

  6. Guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata

    Directory of Open Access Journals (Sweden)

    Won-Gyu Bae

    2015-12-01

    Full Text Available Engineering complex extracellular matrix (ECM is an important challenge for cell and tissue engineering applications as well as for understanding fundamental cell biology. We developed the methodology for fabrication of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with original wrinkling technique for the generation of well-defined native ECM-like platforms by culturing fibroblast cells on the multiscale substrata [1]. This paper provides information on detailed characteristics of polyethylene glycol-diacrylate multiscale substrata. In addition, a possible model for guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata is proposed.

  7. Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G2/M accumulation in cardiac fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Xu Dong LIAO; Xiao Hui WANG; Hai Jing JIN; Lan Ying CHEN; Quan CHEN

    2004-01-01

    Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study,a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes,but not cardiac fibroblasts,underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψm) reduction,indicative of mitochondrial permeability transition pore (PTP)opening. Cyclosporin A,an inhibitor of PTP,inhibited stretch-induced cyto c release,△ψm reduction and apoptosis,suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins,including Bax and Bad,in cardiomyocytes,but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψm reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery,probably by targeting to PTP. Interestingly,the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis,suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore,we showed that p21 was upregulated and cyclin B l was downregulated only in cardiac fibroblasts,which may be associated with G2/M accumulation in response to mechanical stretch.

  8. Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C.

    Science.gov (United States)

    Kim, Tae-im; Tchah, Hungwon; Cho, Eun Hee; Kook, Michael S

    2004-01-01

    To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction

  9. Effects of tanshinone IIA on the transforming growth factor β1/Smad signaling pathway in rat cardiac fibroblasts

    Directory of Open Access Journals (Sweden)

    Cheng-Ye Zhan

    2014-01-01

    Full Text Available Objectives: This study explores the mechanism of tanshinone IIA (TSN-mediated inhibition of myocardial fibrosis by investigating the effect of TSN on transforming growth factor β1 (TGFβ1 signal transduction in rat cardiac fibroblasts (CFs. Materials and Methods: CFs were isolated from neonatal Sprague-Dawley rats by trypsin digestion and differential adhesion and stimulated with 5 ng/mL TGFβ1 and TSN (10−6 , 10−5 , or 10−4 mol/L. The expression of fibronectin (FN mRNA in the CFs was determined using reverse transcriptase-polymerase chain reaction and the protein expression of FN and Smads in CFs was detected using Western blot. The intracellular expression and localization of Smads in the CFs were analyzed using immunocytochemistry. Results: TGFβ1 induced the expression of FN and Smads in a time-dependent manner. At the end of the culture treatment, the mRNA expression of FN and the expression of phosphorylated Smad2/3 (p-Smad2/3 increased significantly (P < 0.01. TSN pretreatment (10−5 and 10−4 mol/L reduced the expression of FN and p-Smad2/3 (P < 0.01 following TGFβ1 stimulation and led to a significant decrease in the nuclear staining intensity and a positive rate of p-Smad2/3 (P < 0.05 and P < 0.01, respectively. Conclusion: The inhibitory effect of TSN on myocardial fibrosis may be associated with its inhibition of TGFβ1-induced Smad2/3 phosphorylation and p-Smad2/3 nuclear translocation, which blocks the TGFβ1/Smad signaling pathway in CFs.

  10. Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

    Directory of Open Access Journals (Sweden)

    Hidetatsu Outani

    Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

  11. Sexual counselling of cardiac patients in Europe: culture matters.

    Science.gov (United States)

    Goossens, E; Norekvål, T M; Faerch, J; Hody, L; Olsen, S S; Darmer, M R; Jaarsma, T; Moons, P

    2011-10-01

    Sexual problems are common amongst cardiac patients, and concerns may arise when resuming sexual activities after a cardiac event. Sexual counselling is therefore indispensible. Culture is an identified barrier to talking about sex, but research is lacking on whether and how culture influences nurses in providing sexual counselling. This cross-sectional descriptive study assessed four areas related to sexual counselling provided by cardiovascular nurses. We investigated the impact of culture on these areas by surveying cardiovascular nurses living in Denmark, Norway and two regions of Belgium - Flanders, Dutch-speaking region and Wallonia, French-speaking region. Overall, 819 participants were recruited as they attended cardiovascular nursing congresses in Denmark, Norway and Belgium. Subjects completed the Undertaking Nursing Interventions Throughout Europe (UNITE) sexual counselling questionnaire, measuring practice, responsibility, confidence and perceived comfort of patients. Controlling for demographic, educational and professional covariates, we performed multiple linear regression analysis to determine the impact of culture on sexual counselling. All four subscale scores were independently associated with culture. Danish nurses counselled patients significantly more often, reported feeling more responsibility and confidence and estimated more comfort in patients than Norwegian, Flemish and Walloon nurses. This study showed that culture matters with respect to sexual counselling for cardiac patients. Interventions should be developed improving sexual counselling of cardiac patients. Educational courses and training of healthcare professionals on sexual counselling should be more sensitive to sociocultural differences. Cross-cultural perspectives may bias attitudes of professionals as they deal with concerns of cardiac patients about resuming sexual activity. © 2011 Blackwell Publishing Ltd.

  12. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid

    Directory of Open Access Journals (Sweden)

    Fernanda Gonçalves BASSO

    Full Text Available Abstract Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  13. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

    Science.gov (United States)

    Basso, Fernanda Gonçalves; Soares, Diana Gabriela; Pansani, Taisa Nogueira; Turrioni, Ana Paula Silveira; Scheffel, Débora Lopes; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-11-28

    Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  14. Cytotoxicity evaluation of root repair materials in human-cultured periodontal ligament fibroblasts

    Directory of Open Access Journals (Sweden)

    Voruganti Samyuktha

    2014-01-01

    Full Text Available Aim: To evaluate the cytotoxicity of three root repair materials, mineral trioxide aggregate (MTA, Endosequence Root Repair Material and Biodentine in human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts were cultured from healthy premolar extracted for orthodontic purpose. Cells in the third passage were used in the study. The cultured fibroblast cells were placed in contact with root repair materials: (a Biodentine, (b MTA, (c Endosequence, (d control. The effects of these three materials on the viability of Periodontal ligament (PDL fibroblasts were determined by trypan blue dye assay after 24 hours and 48-hour time period. Cell viability was determined using inverted phase contrast microscope. Statistical Analysis: Cell viability was compared for all the experimental groups with Wilcoxons matched pair test. Results: At the 24-hour examination period, all the materials showed increased cell viability. At 48-hour time period, there is slight decrease in cell viability. Mineral trioxide aggregate showed statistically significant increase in the cell viability when compared to other root repair materials. Conclusion: Mineral trioxide aggregate was shown to be less toxic to periodontal ligament fibroblasts than Endosequence Root Repair Material and Biodentine.

  15. Cytotoxicity evaluation of root repair materials in human-cultured periodontal ligament fibroblasts

    Science.gov (United States)

    Samyuktha, Voruganti; Ravikumar, Pabbati; Nagesh, Bolla; Ranganathan, K.; Jayaprakash, Thumu; Sayesh, Vemuri

    2014-01-01

    Aim: To evaluate the cytotoxicity of three root repair materials, mineral trioxide aggregate (MTA), Endosequence Root Repair Material and Biodentine in human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts were cultured from healthy premolar extracted for orthodontic purpose. Cells in the third passage were used in the study. The cultured fibroblast cells were placed in contact with root repair materials: (a) Biodentine, (b) MTA, (c) Endosequence, (d) control. The effects of these three materials on the viability of Periodontal ligament (PDL) fibroblasts were determined by trypan blue dye assay after 24 hours and 48-hour time period. Cell viability was determined using inverted phase contrast microscope. Statistical Analysis: Cell viability was compared for all the experimental groups with Wilcoxons matched pair test. Results: At the 24-hour examination period, all the materials showed increased cell viability. At 48-hour time period, there is slight decrease in cell viability. Mineral trioxide aggregate showed statistically significant increase in the cell viability when compared to other root repair materials. Conclusion: Mineral trioxide aggregate was shown to be less toxic to periodontal ligament fibroblasts than Endosequence Root Repair Material and Biodentine. PMID:25298650

  16. Apoptotic markers in cultured fibroblasts correlate with brain metabolites and regional brain volume in antipsychotic-naive first-episode schizophrenia and healthy controls

    NARCIS (Netherlands)

    Batalla, A.; Bargallo, N.; Gasso, P.; Molina, O.; Pareto, D.; Mas, S.; Roca, J.M.; Bernardo, M.; Lafuente, A.; Parellada, E.

    2015-01-01

    Cultured fibroblasts from first-episode schizophrenia patients (FES) have shown increased susceptibility to apoptosis, which may be related to glutamate dysfunction and progressive neuroanatomical changes. Here we determine whether apoptotic markers obtained from cultured fibroblasts in FES and cont

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-02-01

    The authors evaluated the interaction of (/sub 3/H)1,25(OH)/sup 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)/sub 2/D (1,25(OH)/sub 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 (/sub 3/H)1,25(OH)/sup 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 (/sub 3/H)1,25(OH)/sup 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 (/sub 3/H)1,25(OH)/sup 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)/sup 2/D resulted from five or six distinct genetic mutations in six kindreds.

  18. Microfluidic cardiac cell culture model (μCCCM).

    Science.gov (United States)

    Giridharan, Guruprasad A; Nguyen, Mai-Dung; Estrada, Rosendo; Parichehreh, Vahidreza; Hamid, Tariq; Ismahil, Mohamed Ameen; Prabhu, Sumanth D; Sethu, Palaniappan

    2010-09-15

    Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (μCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

  19. Adenylyl cyclase type 6 overexpression selectively enhances beta-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts.

    Science.gov (United States)

    Liu, Xiaoqiu; Thangavel, Muthusamy; Sun, Shu Qiang; Kaminsky, Joseph; Mahautmr, Penden; Stitham, Jeremiah; Hwa, John; Ostrom, Rennolds S

    2008-06-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP2 (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP2 receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.

  20. Carbon Nanohorns Promote Maturation of Neonatal Rat Ventricular Myocytes and Inhibit Proliferation of Cardiac Fibroblasts: a Promising Scaffold for Cardiac Tissue Engineering

    Science.gov (United States)

    Wu, Yujing; Shi, Xiaoli; Li, Yi; Tian, Lei; Bai, Rui; Wei, Yujie; Han, Dong; Liu, Huiliang; Xu, Jianxun

    2016-06-01

    Cardiac tissue engineering (CTE) has developed rapidly, but a great challenge remains in finding practical scaffold materials for the construction of engineered cardiac tissues. Carbon nanohorns (CNHs) may be a potential candidate due to their special structure and properties. The purpose of this study was to assess the effect of CNHs on the biological behavior of neonatal rat ventricular myocytes (NRVMs) for CTE applications. CNHs were incorporated into collagen to form growth substrates for NRVMs. Transmission electron microscopy (TEM) observations demonstrated that CNHs exhibited a good affinity to collagen. Moreover, it was found that CNH-embedded substrates enhanced adhesion and proliferation of NRVMs. Immunohistochemical staining, western blot analysis, and intracellular calcium transient measurements indicated that the addition of CNHs significantly increased the expression and maturation of electrical and mechanical proteins (connexin-43 and N-cadherin). Bromodeoxyuridine staining and a Cell Counting Kit-8 assay showed that CNHs have the ability to inhibit the proliferation of cardiac fibroblasts. These findings suggest that CNHs can have a valuable effect on the construction of engineered cardiac tissues and may be a promising scaffold for CTE.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-02-01

    We evaluated the interaction of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)/sub 2/D (1,25(OH)/sub 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 (/sup 3/H)1,25(OH)/sub 2/D/sub 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 (/sup 3/H)1,25(OH)/sub 2/D/sub 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)/sub 2/D resulted from five or six distinct genetic mutations in six kindreds.

  2. Effect of three commercial mouth rinses on cultured human gingival fibroblast: An in vitro study

    Directory of Open Access Journals (Sweden)

    Flemingson

    2008-01-01

    Full Text Available Aim: To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1% upon cultured human gingival fibroblast proliferation. Materials and Methods: Human gingival fibroblasts were cultured and incubated in Dulbecco′s Minimum Eagle′s Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution at 37°C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco′s Minimum Eagle′s Medium without adding mouth rinses, for similar duration of exposure at 37°C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h. Results: In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine. Conclusion: These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.

  3. Dupuytren's disease: comparative growth dynamics and morphology between cultured myofibroblasts (nodule) and fibroblasts (cord).

    Science.gov (United States)

    Vande Berg, J S; Gelberman, R H; Rudolph, R; Johnson, D; Sicurello, P

    1984-01-01

    The excised palmar fascia of 11 patients with Dupuytren's disease was separated clinically into nodules and cords. Myofibroblasts were seen by light and electron microscopy in each of the nodules, but the cords generally lacked myofibroblasts. Only one cord specimen had microscopic features that were intermediate between nodule and cord. Electron microscopy demonstrated that in vivo differences between myofibroblasts from nodules and fibroblasts from cords and control skin samples could be preserved in vitro. Growth studies showed slower growth of cultured myofibroblasts (mean +/- SD generation time 68.7 +/- 15 h) than cord-derived fibroblasts (mean +/- SD generation time 51.5 +/- 0.9 h). These data suggest that the life cycle of the myofibroblasts from Dupuytren's disease nodules differs from that of fibroblasts found in cordlike tissues. These myofibroblasts have biological characteristics nearly identical to those of myofibroblasts found in other contracting tissues, such as granulating wounds and breast cancer.

  4. Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

    DEFF Research Database (Denmark)

    Jeppesen, Pia Lindgren; Christensen, Gitte Lund; Schneider, Mikael

    2011-01-01

    Background and purpose: The Angiotensin II type 1 receptor (AT(1) R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we asked whether miRNAs might...

  5. Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts: a possible defense against transforming growth factor-ß mediated fibrosis

    LENUS (Irish Health Repository)

    Watson, Chris J

    2012-07-07

    AbstractBackgroundMechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts’ response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.Methods and resultsThe effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.ConclusionWe postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

  6. Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan

    Science.gov (United States)

    Patrick, Alison; Seluanov, Michael; Hwang, Chaewon; Tam, Jonathan; Khan, Tanya; Morgenstern, Ari; Wiener, Lauren; Vazquez, Juan M.; Zafar, Hiba; Wen, Robert; Muratkalyeva, Malika; Doerig, Katherine; Zagorulya, Maria; Cole, Lauren; Catalano, Sophia; Lobo Ladd, Aliny AB; Coppi, A. Augusto; Coşkun, Yüksel; Tian, Xiao; Ablaeva, Julia; Nevo, Eviatar; Gladyshev, Vadim N.; Zhang, Zhengdong D.; Vijg, Jan; Seluanov, Andrei; Gorbunova, Vera

    2016-01-01

    Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan. PMID:27163160

  7. Dexamethasone inhibition of IL-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Lu, Ying; Fukuda, Ken; Liu, Yang; Kumagai, Naoki; Nishida, Teruo

    2004-09-01

    Corticosteroids regulate the functions of inflammatory cells. The purpose of the present study was to investigate the effect of dexamethasone on collagen degradation by corneal fibroblasts, an underlying cause of corneal ulceration. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of IL-1beta or dexamethasone. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was evaluated by immunoblot analysis, gelatin zymography, and reverse transcription and real-time polymerase chain reaction. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was assessed by immunoblot analysis. Dexamethasone inhibited IL-1beta-induced collagen degradation by corneal fibroblasts in a dose-dependent manner. Both the synthesis and activation of MMPs and the expression of TIMPs were inhibited by dexamethasone, as was the activity of plasmin in culture supernatants. Dexamethasone also inhibited the IL-1beta-induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not that of p38. Dexamethasone exerted multiple effects on the MMP-TIMP system in corneal fibroblasts and thereby inhibited IL-1beta-induced collagen degradation by these cells. Inhibition of the IL-1beta-induced activation of ERK and JNK may contribute to these effects of dexamethasone. Copyright Association for Research in Vision and Ophthalmology

  8. An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.

    Directory of Open Access Journals (Sweden)

    Tokiwa,Takayoshi

    1986-04-01

    Full Text Available The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF, which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC or putrescine (PUT. The growth of HuF cells was inhibited by HC at a concentration of 10(-2 M and by PUT at a concentration higher than 10(-3 M. Long-term treatment of HuF cells with 10(-3 M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5M to 10(-3 M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3 M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

  9. Biocompatibility of three bioabsorbable membranes assessed in FGH fibroblasts and human osteoblast like cells culture.

    Science.gov (United States)

    Soares, Michelle Pereira Costa Mundim; Soares, Paulo Vinícius; Pereira, Analice Giovani; Moura, Camilla Christian Gomes; Soares, Priscila Barbosa Ferreira; Naves, Lucas Zago; de Magalhães, Denildo

    2014-08-06

    Specific physical and chemical features of the membranes may influence the healing of periodontal tissues after guided tissue regeneration (GTR). The aim of the present investigation was to analyze the biological effects of three bioabsorbable membranes. The hypothesis is that all tested membranes present similar biological effects. Human osteoblast like-cells (SaOs-2) and gingival fibroblasts FGH (BCRJ -RJ) were cultured in DMEM medium. The viability of the cells cultured on the membranes was assesses using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Quantitative determination of activated human Transforming Growth Factor beta 1 (TGF-β1) on the supernatants of the cell culture was observed. Samples were examined using scanning electron microscope (SEM). SaOs2, in 24 hours, PLA group showed higher values when compared to other groups (P statistical significance values when compared two times. In 4 h and 24 h, for the fibroblasts group, significantly difference was found to PLA membrane, when compared with the other groups (p statistically significant difference (p analysis of culture supernatants of fibroblasts, in 24 hours, only PLA group presented significant difference (p = 0,008). The biomaterials analyzed did not show cytotoxicity, since no membrane presented lower results than the control group. PLA membrane presented the best performance due to its higher cell viability and absorbance levels of proliferation. Both collagen membranes showed similar results either when compared to each other or to the control group.

  10. Characterization of perceived hyperoxia in isolated primary cardiac fibroblasts and in the reoxygenated heart.

    Science.gov (United States)

    Roy, Sashwati; Khanna, Savita; Wallace, William A; Lappalainen, Jani; Rink, Cameron; Cardounel, Arturo J; Zweier, Jay L; Sen, Chandan K

    2003-11-21

    Under normoxic conditions, pO2 ranges from 90 to Bierl, M., Pendyala, S., Levy, D., Sharma, N., Venojarvi, M., Strauch, A., Orosz, C. G., and Sen, C. K. (2003) Circ. Res. 92, 264-271). Here, we sought to characterize the genomic response to perceived hyperoxia in CF using GeneChips trade mark. Candidate genes were identified, confirmed and clustered. Cell cycle- and differentiation-associated genes represented a key target of perceived hyperoxia. Bioinformatics-assisted pathway reconstruction revealed the specific signaling processes that were sensitive to perceived hyperoxia. To test the significance of our in vitro findings, a survival model of rat heart focal ischemia-reperfusion (I-R) was investigated. A significant induction in p21 mRNA expression was observed in I-R tissue. The current results provide a comprehensive molecular definition of perceived hyperoxia in cultured CF. Furthermore, the first evidence demonstrating activation of perceived hyperoxia sensitive genes in the cardiac I-R tissue is presented.

  11. Cardiac Fibroblast-Specific Activating Transcription Factor 3 Protects Against Heart Failure by Suppressing MAP2K3-p38 Signaling.

    Science.gov (United States)

    Li, Yulin; Li, Zhenya; Zhang, Congcong; Li, Ping; Wu, Yina; Wang, Chunxiao; Lau, Wayne Bond; Ma, Xin-Liang; Du, Jie

    2017-03-01

    Background -Hypertensive ventricular remodeling is a common cause of heart failure. However, the molecular mechanisms regulating ventricular remodeling remain poorly understood. Methods -We used a discovery-driven/nonbiased approach to indentify increased ATF3 expression in hypertensive heart. We employed loss/gain of function approaches to understand the role of ATF3 in heart failure. We also examine the mechanisms through transcriptome, CHIP-seq analysis and in vivo and vitro experiments. Results -ATF3 expression increased in murine hypertensive heart and human hypertrophic heart. Cardiac fibroblast cells are the primary cell type expressing high ATF3 levels in response to hypertensive stimuli. ATF3 knockout (ATF3KO) markedly exaggerated hypertensive ventricular remodeling, a state rescued by lentivirus-mediated/miRNA-aided cardiac fibroblast-selective ATF3 overexpression. Conversely, conditional cardiac fibroblast cell-specific ATF3 transgenic overexpression significantly ameliorated ventricular remodeling and heart failure. We identified Map2K3 as a novel ATF3 target. ATF3 binds with the Map2K3 promoter, recruiting HDAC1, resulting in Map2K3 gene-associated histone deacetylation, thereby inhibiting Map2K3 expression. Genetic Map2K3 knockdown rescued the pro-fibrotic/hypertrophic phenotype in ATF3KO cells. Finally, we demonstrated that p38 is the downstream molecule of Map2K3 mediating the pro-fibrotic/hypertrophic effects in ATF3KO animals. Inhibition of p38 signaling reduced TGF-β signaling-related pro-fibrotic and hypertrophic gene expression, and blocked exaggerated cardiac remodeling in ATF3KO cells. Conclusions -Our study provides the first evidence that ATF3 upregulation in cardiac fibroblasts in response to hypertensive stimuli protects heart by suppressing Map2K3 expression and subsequent p38-TGF-β signaling. These results suggest that positive modulation of cardiac fibroblast ATF3 may represent a novel therapeutic approach against hypertensive

  12. Teratogenic and cytotoxic effects of VOsalen complex on chicken embryos, hepatic and fibroblastic- cell cultures

    Directory of Open Access Journals (Sweden)

    Abdolmaleki A

    2013-04-01

    Full Text Available Background: Salen metal complexes are used successfully in a wide range of asymmet-ric reactions and important in the pharmaceutical and industry. On the toxicity of salen vanadium oxide (VOsalen on embryo and cell cultures, little information is available. In the present study, the toxic and teratogenic effects of VOsalen was evaluated against chicken embryos as a animal model and liver and fibroblast cell cultures which was derived from the embryo.Methods: The VOsalen compound was synthesized. The compound solution was inject-ed in triplicate examination, in the air sac of the eggs, at third day of incubation. Treat-ed and control eggs, on day 19 of incubation opened and embryos were weighted, then mortality rate was recorded. The liver and fibroblast cell culture were treated by this and survival fraction was recorded.Results: The survived fraction of the embryos depends on the compound concentration. In concentration of 300μM/egg, 36/32% of the embryos survived and the Lethal dose 50% (LD50 was 226/37 μM/egg. Morphological study of the treated embryos showed retarded growth, and skeletal staining showed the deletion of caudal vertebrate. The compound was inhibited liver and fibroblast cells growth with IC50 1047/25 and 1036/82μM respectively. The cytoplasm of treated cells became dense and their interco-nnections were loosed.Conclusion: The VOsalen compound had low toxic effects against the embryos and the cultured cells at the concentrations. Significant cytotoxic effect was not observed in the treated cells. However the proliferative cells were affected significantly in comparison with the cells which their growth was stopped. The effect of VOsalen compound against replication of liver cells were lower than fibroblast cells.

  13. Metformin inhibits aldosterone-induced cardiac fibroblast activation, migration and proliferation in vitro, and reverses aldosterone+salt-induced cardiac fibrosis in vivo.

    Science.gov (United States)

    Mummidi, Srinivas; Das, Nitin A; Carpenter, Andrea J; Kandikattu, Hemanthkumar; Krenz, Maike; Siebenlist, Ulrich; Valente, Anthony J; Chandrasekar, Bysani

    2016-09-01

    The overall goals of this study were to investigate whether metformin exerts anti-fibrotic effects in aldosterone (Aldo)+salt-treated wild type mouse hearts, and determine the underlying molecular mechanisms in isolated adult cardiac fibroblasts (CF). In vitro, Aldo induced CF activation, migration, and proliferation, and these effects were inhibited by metformin. Further, Aldo induced PPM1A (Protein Phosphatase Magnesium Dependent 1A) activation and inhibited AMPK phosphorylation. At a pharmacologically relevant concentration, metformin restored AMPK activation, and inhibited Aldo-induced Nox4/H2O2-dependent TRAF3IP2 induction, pro-inflammatory cytokine expression, and CF migration and proliferation. Further, metformin potentiated the inhibitory effects of spironolactone, a mineralocorticoid receptor antagonist, on Aldo-induced collagen expression, and CF migration and proliferation. These results were recapitulated in vivo, where metformin reversed Aldo+salt-induced oxidative stress, suppression of AMPK activation, TRAF3IP2 induction, pro-inflammatory cytokine expression, and cardiac fibrosis, without significantly modulating systolic blood pressure. These in vitro and in vivo data indicate that metformin has the potential to reduce adverse cardiac remodeling in hypertensive heart disease.

  14. [Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts].

    Science.gov (United States)

    Wu, Qi'er; Lyu, Lu; Xin, Haiming; Luo, Liang; Tong, Yalin; Mo, Yongliang; Yue, Yigang

    2016-06-01

    To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post

  15. Experimental study on He- Ne laser irradiation to inhibit scar fibroblast growth in culture

    Institute of Scientific and Technical Information of China (English)

    舒彬; 吴宗耀; 郝林林; 曾登芬; 冯光锐; 林永辉

    2002-01-01

    To explore the inhibitory effect of He-Ne laser irradiation on fibroblast growth of hypertrophic scars in culture. Methods: He-Ne laser with wavelength of 632.8 nm,power density of 50 mW/cm2 and doses of 3 J/cm2,30 J/cm2, 90 J/cm2 and 180 J/cm2 was used to irradiate human scar fibroblasts in culture 1, 3 and 5 times respectively, and then the cell count and cell cycle analysis were done. Results: Repeated irradiation with He-Ne laser at dose of 180 J/cm2 three and five times led to an evident decrease in total cell number compared with that of the control group and there was a significant difference ( P <0.05). The cell cycle analysis showed after three and five times of irradiation with 180 J/cm2 He-Ne laser the cell number in S-phase decreased from 51% to 20% and 14% respectively, the cell number in G0/G1 phase increased from 28% to 55% and 60% respectively, and the cell percentage in Sub-G1 phase was 6.7% and 9.8% respectively. Conclusions: Repeated irradiation with 180 J/cm2 He-Ne laser can inhibit scar fibroblasts growth in culture.It may be that He-Ne laser irradiation causes cell stagnation in G0/G1 phase and apoptosis.

  16. Low-Dose UVA Radiation-Induced Adaptive Response in Cultured Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhongrong Liu

    2012-01-01

    Full Text Available Objective. To investigate the mechanism of the adaptive response induced by low-dose ultraviolet A (UVA radiation. Methods. Cultured dermal fibroblasts were irradiated by a lethal dose of UVA (86.4 J/cm2 with preirradiation of single or repetitive low dose of UVA (7.2 J/cm2. Alterations of cellular morphology were observed by light microscope and electron microscope. Cell cycle and cellular apoptosis were assayed by flow cytometer. The extent of DNA damage was determined by single-cell gel electrophoresis (SCGE. Results. The cultured dermal fibroblasts, with pretreatment of single or repetitive irradiation of 7.2 J/cm2 UVA relieved toxic reaction of cellular morphology and arrest of cell cycle, decreased apoptosis ratio, reduced DNA chain breakage, and accelerated DNA repair caused by subsequent 86.4 J/cm2 UVA irradiation. Compared with nonpretreatment groups, all those differences were significant (P<0.01 or P<0.05. Conclusions. The adaptation reaction might depend on the accumulated dose of low-dose UVA irradiation. Low-dose UVA radiation might induce adaptive response that may protect cultured dermal fibroblasts from the subsequent challenged dose of UVA damage. The duration and protective capability of the adaptive reaction might be related to the accumulated dose of low-dose UVA Irradiation.

  17. Cartilage Intermediate Layer Protein 1 Suppresses TGF-β Signaling in Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Kazuhiro Shindo

    2017-06-01

    Conclusion: We identified CILP1 as a potential regulator of cardiac fibrosis by inhibiting TGF-β signaling, and these results suggest the promise of CILP1 as a novel therapeutic target for preventing cardiac fibrosis and heart failure in MI patients.

  18. Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

    Directory of Open Access Journals (Sweden)

    Ertl Ronald F

    2001-09-01

    Full Text Available Abstract Background Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. Methods Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Results Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P P P 2 production was significantly increased by co-culture and its presence attenuated collagen degradation. Conclusion The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

  19. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  20. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

    Science.gov (United States)

    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  1. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

    Science.gov (United States)

    Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

    2012-12-01

    The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

  2. Fibulin-6 regulates pro-fibrotic TGF-β responses in neonatal mouse ventricular cardiac fibroblasts.

    Science.gov (United States)

    Chowdhury, Arpita; Hasselbach, Lisa; Echtermeyer, Frank; Jyotsana, Nidhi; Theilmeier, Gregor; Herzog, Christine

    2017-02-17

    Fibulin-6, an essential component of extracellular matrix determines the architecture of cellular junctions in tissues undergoing strain. Increased expression and deposition of fibulin-6 facilitates fibroblast migration in response to TGF-β, following myocardial infarction in mouse heart. The underlying mechanism still remains elusive. In conjunction with our previous study, we have now demonstrated that in fibulin-6 knockdown (KD) fibroblasts, not only TGF-β dependent migration, but also stress fiber formation, cellular networking and subsequently fibroblast wound contraction is almost abrogated. SMAD dependent TGF-β pathway shows ~75% decreased translocation of R-SMAD and co-SMAD into the nucleus upon fibulin-6 KD. Consequently, SMAD dependent pro-fibrotic gene expression is considerably down regulated to basal levels both in mRNA and protein. Also, investigating the non-SMAD pathways we observed a constitutive increase in pERK-levels in fibulin-6 KD fibroblast compared to control, but no change was seen in pAKT. Immunoprecipitation studies revealed 60% reduced interaction of TGF-β receptor II and I (TGFRII and I) accompanied by diminished phosphorylation of TGFRI at serin165 in fibulin-6 KD cells. In conclusion, fibulin-6 plays an important role in regulating TGF-β mediated responses, by modulating TGF-β receptor dimerization and activation to further trigger downstream pathways.

  3. Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age.

    Science.gov (United States)

    Ivanov, Nikolay A; Tao, Ran; Chenoweth, Joshua G; Brandtjen, Anna; Mighdoll, Michelle I; Genova, John D; McKay, Ronald D; Jia, Yankai; Weinberger, Daniel R; Kleinman, Joel E; Hyde, Thomas M; Jaffe, Andrew E

    2016-02-01

    Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), "block" (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts-83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation--while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p = 0

  4. Comparative Culturing of 3T3 Swiss J2 Mouse Embryo Fibroblasts on Modified Chitosan Matrices.

    Science.gov (United States)

    Alekhin, A I; Gaenko, G P

    2016-07-01

    Comparative culturing of mouse embryo fibroblasts on chitosan matrices and culture plastic was carried out. During the first 2 h of culturing (lag phase), cell adhesion to chitosan and chitosan-gelatin matrices was 20-30% higher than adhesion to culture plastic (control). During the stationary phase, 80% cells adhered to chitosan matrices (vs. 60% in the control). Cell culturing on chitosan matrices was carried out without medium replacement with fresh portions. The cells remained viable within 5 days of culturing. Cell death phase was observed on day 6 of culturing on chitosan matrices: cell adhesion dropped to 50%. Culturing on culture plastic was carried out with daily medium replacement with a fresh portion. Cell death phase (50% decrease in the number of adherent cell) under these condition was observed on day 5. It seems that the observed effect was a result of contact interactions of cell integrins and chitosan ligands, modulation of transmembrane signal, eventually modifying the expression of cell genes. This effect can be required in regenerative medicine for production of primary cell culture.

  5. Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  6. Effects of Balcalin on Expression of Inducible Nitric Oxide Synthase In Cultured Fibroblasts Stimulated by Cytokines

    Institute of Scientific and Technical Information of China (English)

    毕新岭; 顾军; 聂本勇; 李泉; 刘辉; 米庆胜

    2004-01-01

    Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α((TNF-α), interferon-γ(IFN-γ), interleukin-8 (IL-8) in different groups, iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results. Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1.0×106 U/L TNF-α, 0.2×106U/L IFN-γ, 0.2×106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50μg/ ml of baicalin. Conclusion. Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.

  7. Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture

    Energy Technology Data Exchange (ETDEWEB)

    Kolodny, G.M.

    1975-01-01

    Growing and confluent cultures of mouse fibroblasts were labeled with /sup 3/H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.

  8. Simultaneous isolation of myocardial cells and cardiac fibroblasts from neonatal rats%新生大鼠心肌细胞和心肌成纤维细胞的同时分离

    Institute of Scientific and Technical Information of China (English)

    施雨露; 李晓辕; 曹美娜; 于姝媛; 王苹

    2013-01-01

      背景:利用心肌组织中不同细胞的黏附特性差异,通过优化心肌细胞的培养条件,一次分离同时获得心肌和心肌成纤维细胞。目的:探讨新生大鼠心肌和心肌成纤维细胞同时分离的有效方法。方法:采用低浓度胰蛋白酶冷消化结合胶原酶复合消化,差速分离心肌和心肌成纤维细胞。通过使用不同种类的血清改良心肌细胞培养条件,免疫荧光鉴定心肌细胞和心肌成纤维细胞纯度。结果与结论:心肌培养48 h时Troponin T阳性细胞率为89.3%。第2代的心肌成纤维细胞培养波形蛋白阳性细胞率为93.6%。含马血清的培养基组心肌细胞阳性率明显高于牛血清培养组(P <0.05)。实验应用的新生大鼠心肌细胞分离培养方法,心肌细胞和心肌成纤维细胞纯度高、活性好,且能同时获得心肌细胞和心肌成纤维细胞。%  BACKGROUND: Myocardial cel s and cardiac fibroblasts are simultaneously isolated through optimizing the culture conditions of myocardial cel s, by use of the variations of adhesion properties of myocardial cel s. OBJECTIVE: To explore the co-separation method for myocardial cel s and cardiac fibroblasts in newborn rats. METHODS: Myocardial tissue in newborn rats was digested with low concentration trypsin at cold conditions and with col agenase II at 37 ℃. The myocardial cel s and cardiac fibroblasts were isolated with differential centrifugation method, and myocardial cel culture conditions were improved using different kinds of serum. Immunofluorescence staining was used to evaluate the purity of myocardial cel s and cardiac fibroblasts. RESULTS AND CONCLUSION: The myocardial Troponin T positive cel rate was 89.3% at culture 48 hours, and myocardial fibroblast vimentin positive cel rate was 93.6% at passage 2. The percentage of positive myocardial cel s cultured with medium containing horse serum was obviously higher than that with bovine serum

  9. Comparative use of glucose and fructose in cultured fibroblasts from patients with hereditary fructose intolerance.

    Science.gov (United States)

    Lemonnier, F; Delhotal-Landes, B; Couturier, M; Decimo, D; Odiévre, M; Gautier, M; Lemonnier, A

    1987-01-01

    The utilization of fructose and glucose by fibroblast cultures obtained from patients with hereditary fructose intolerance (HFI) was studied in comparison with fibroblast controls. The cell growth, the time course of D-glucose or D-fructose uptake and the consumption of fructose were similar for both HFI and control cells. Some results showed significant differences between these two cell types: HFI cells consumed less glucose, produced less lactate and contained less glycogen than control cells. Furthermore, significantly less [U-14C]D-glucose and [U-14C]D-fructose was incorporated into lipids in HFI cells than in control cells. The mechanisms responsible for these differences observed between the two cell types are not known.

  10. Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

    Directory of Open Access Journals (Sweden)

    Claudia Casnici

    2014-01-01

    Full Text Available The composition of synovial fluid in rheumatoid arthritis (RA is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, “in vitro” studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform “in vitro” studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in “in vitro” studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

  11. Inhibitory Effects of Enalaprilat on Rat Cardiac Fibroblast Proliferation via ROS/P38MAPK/TGF-β1 Signaling Pathway

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    Du-Juan Yu

    2012-03-01

    Full Text Available Enalaprilat (Ena., an angiotensin II (Ang II converting enzyme inhibitor (ACEI, can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS; immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK and transforming growth factor-β1 (TGF-β1 protein expression, respectively. The results showed that Ang II (10–7 M stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant, SB203580 (a p38MAPK inhibitor or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group, as well as an elevation of TGF-β1 meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-β1 protein expression in rats’ CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-β1 signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.

  12. Zinc and propolis reduces cytotoxicity and proliferation in skin fibroblast cell culture: total polyphenol content and antioxidant capacity of propolis.

    Science.gov (United States)

    Tyszka-Czochara, Małgorzata; Paśko, Paweł; Reczyński, Witold; Szlósarczyk, Marek; Bystrowska, Beata; Opoka, Włodzimierz

    2014-07-01

    It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees' products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01% (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01% (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts' viability, although propolis revealed its antiproliferative action and caused mild necrosis.

  13. Effect of Qiangxin Decoction on Cardiac Fibroblast Proliferation and Collagen Synthesis%中药强心饮含药血清对心肌成纤维细胞增殖和胶原合成的影响

    Institute of Scientific and Technical Information of China (English)

    沈雁; 严世芸

    2011-01-01

    目的:研究强心饮对肿瘤坏死因子α(tumor necrosis factor α,TNF-α)诱导新生大鼠心肌成纤维细胞(cardi-ac fibroblasts CFs)增殖和胶原合成的影响,探讨其抑制心肌纤维化的作用机制.方法:采用胰酶消化法分离培养乳鼠CFs,建立TNF-α诱导新生大鼠CFs纤维化病理模型,分别采用四氮唑蓝(MTT)比色法、羟脯氨酸法检测不同浓度强心饮含药血清对CFs增殖和胶原含量影响.结果:TNF-α能够刺激CFs增殖和胶原合成,与空白组相比差异显著(P<0.05);强心饮抑制TNF-α诱导CFs增殖和胶原合成(P均<0.05).结论:强心饮通过抑制TNF-α诱导的CFs增殖和胶原合成,发挥有效的抗心肌纤雏化作用.%Objective:To investigate the effect of Qiangxin Decoction on the proliferation and collagen synthesis in neonatal rats cardiac fibroblast(CFs) induced by tumor necrosis factor α (TNF -α), and to explore the mechanism of Qiangxin Decoction on reversing cardiac fibrosis. Methods: Cardiac fibroblasts derived from neonatal rots were cultured with enzymatic dissociation, and CFs pathological model was induced by tumor necrosis factor α ( TNF - α). Thiazolyl blue MTT assay and hydroxyproline were employed to measure Cfs proliferation and collagen synthesis in order to determine the effect of different dosage decoction on CFs proliferation and collagen synthesis. Results :Compare with control group, TNF-α could induce cardiac fibroblast proliferation and increase collagen synthesis significantly ( P < 0. 05 ), while Qiangxin Decoction inhibit Cfs proliferation and collagen synthesis ( P < 0. 05 ). Conclusion: Qiangxin Decoction could reverse cardiac fibrosis, which may be related to inhibit CFs proliferation and collage synthesis induced by TNF -α.

  14. Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

    Science.gov (United States)

    Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2011-01-01

    Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

  15. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    Science.gov (United States)

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili

    2016-04-01

    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  16. Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing.

    Science.gov (United States)

    Loo, Alvin Eng Kiat; Halliwell, Barry

    2012-06-29

    Recently, there has been renewed interest in the role of reactive oxygen species (ROS), especially H(2)O(2), in wound healing. We previously showed that H(2)O(2) stimulates healing in a keratinocyte scratch wound model. In this paper, we used a more complex and physiologically relevant model that involves co-culturing primary keratinocytes and fibroblasts. We found that the two main cell types within the skin have different sensitivities to H(2)O(2) and to the widely used "antioxidant"N-acetyl-l-cysteine (NAC). Keratinocytes were very resistant to the toxicity of H(2)O(2) (250 and 500 μM) or NAC (5 mM). However, the viability of fibroblasts was decreased by both compounds. Using the co-culture model, we also found that H(2)O(2) increases re-epithelialization while NAC retards it. Our data further illustrate the possible role of ROS in wound healing and the co-culture model should be useful for screening agents that may influence the wound healing process. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    Science.gov (United States)

    Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

    1992-01-01

    The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

  18. Accumulation of phosphorus compounds in tissues and cultured skin fibroblasts in patients with hypophosphatasia

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Y.; Tada, Y.

    1988-08-15

    Patients with hypophosphatasia caused by a deficiency of alkaline phosphatase first showed marked accumulation of phosphoethanolamine and other phosphorus compounds in kidney and liver, while in placenta and intestine contents of these compounds were within a normal range. Furthermore, /sup 32/P-incorporation in cultured skin fibroblasts of patients with hypophosphatasia was increased about two to three times of control. FPLC chromatographic analysis also indicates that the accumulated phosphorus compounds in hypophosphatasia was smaller molecular phosphorus containing compounds. These data provide new pathophysiological aspect of hypophosphatasia.

  19. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors.

    Science.gov (United States)

    Huang, H M; Gibson, G E

    1993-07-15

    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

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

    Science.gov (United States)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon. (c) 2010 Elsevier Ltd. All rights reserved.

  1. Pacemaker interactions induce reentrant wave dynamics in engineered cardiac culture

    Science.gov (United States)

    Borek, Bartłomiej; Shajahan, T. K.; Gabriels, James; Hodge, Alex; Glass, Leon; Shrier, Alvin

    2012-09-01

    Pacemaker interactions can lead to complex wave dynamics seen in certain types of cardiac arrhythmias. We use experimental and mathematical models of pacemakers in heterogeneous excitable media to investigate how pacemaker interactions can be a mechanism for wave break and reentrant wave dynamics. Embryonic chick ventricular cells are cultured invitro so as to create a dominant central pacemaker site that entrains other pacemakers in the medium. Exposure of those cultures to a potassium channel blocker, E-4031, leads to emergence of peripheral pacemakers that compete with each other and with the central pacemaker. Waves emitted by faster pacemakers break up over the slower pacemaker to form reentrant waves. Similar dynamics are observed in a modified FitzHugh-Nagumo model of heterogeneous excitable media with two distinct sites of pacemaking. These findings elucidate a mechanism of pacemaker-induced reentry in excitable media.

  2. The chalcone butein from Rhus verniciflua Stokes inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts

    Directory of Open Access Journals (Sweden)

    Tan Jenny

    2005-03-01

    Full Text Available Abstract Background Butein (3,4,2',4'-tetrahydroxychalone, a plant polyphenol, is a major biologically active component of the stems of Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro. Methods We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL or various other modulators of fibroblast function (troglitazone-1 μg/mL; GW9662-1 μM; meloxican-1 μM; and 3,4 dehydroproline-10 μg/mL. In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL. Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL. Results Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also

  3. Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Karlinsky, J.B.; Goldstein, R.H. (Boston Univ. School of Medicine, MA (USA))

    1989-08-01

    Prostaglandin E2 (PGE2) has been shown to increase the synthesis of hyaluronic acid in cultured fibroblasts by increasing the activity of hyaluronate synthetase, a group of plasma membrane-bound synthetic enzymes. We examined whether PGE2 also increased the activity of those enzyme systems involved in the synthesis of sulfated glycosaminoglycan in the human embryonic lung fibroblast. Exposure of cells to PGE2 resulted in dose-dependent increases in glucosamine incorporation into all sulfated glycosaminoglycan subtypes. PGE2 at 10(-7) mol/L increased total glycosaminoglycan per dish to 21.6 +/- 3.1 micrograms versus 12.0 +/- 2.5 micrograms in control untreated cultures. Stimulation of endogenous PGE2 production by bradykinin had a similar effect on glycosaminoglycan synthesis. To examine whether PGE2 affected sulfated glycosaminoglycan protein core production, cells were labeled with tritiated glucosamine in the presence of cycloheximide. Under these conditions, incorporation of radiolabel into all glycosaminoglycan subtypes was reduced. However, when exogenous sulfated glycosaminoglycan chain initiator (p-nitrophenyl beta-D-xyloside) was added, incorporation of tritiated glucosamine into sulfated glycosaminoglycan increased but not to levels found in control cultures. Application of PGE2 to cultures treated with cycloheximide alone, or to cultures treated with cycloheximide plus xyloside, increased tritiated glucosamine incorporation into chondroitin, dermatan sulfate, and to a lesser extent into heparan sulfate. We conclude that PGE2 stimulates synthesis of all sulfated glycosaminoglycan even in the absence of new protein core production, probably by increasing activities of sulfated glycosaminoglycan synthetase enzymes. PGE2 stimulation of heparan sulfate synthesis is partially dependent on the availability of heparan sulfate-specific protein core.

  4. Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

    Science.gov (United States)

    Fleischmajer, R; MacDonald, E D; Contard, P; Perlish, J S

    1993-09-01

    Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

  5. Ciliated cultured dermal fibroblasts in a patient with hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome.

    Science.gov (United States)

    Haust, M D

    1995-10-01

    Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH)-syndrome is a rare autosomal recessive disorder of the urea cycle, probably caused by a defect in ornithine transport across the hepatic inner mitochondrial membrane. Single rudimentary cilia were present in approximately ten percent of post-divisional or dividing fibroblasts cultured from the skin of a patient with the HHH-syndrome, whereas no such organelles were observed in dermal fibroblasts cultured from normal controls. Since single rudimentary ("primary," "oligo," "solitary") cilia have been observed in a variety of cells in animals and men but the stimuli for their formation and their significance remain controversial, a brief report on their presence in the as yet unreported condition (HHH-syndrome) was considered of interest; hopefully, it might contribute to the ultimate unravelling of some of the unresolved problems. It is of note that unlike the author's previous findings of these unusual organelles in cells affected by a pathological process (atherosclerosis), the rudimentary cilia were observed in the present instance in dividing or postdivisional cells. The implications of these (and other) observations must await further work.

  6. Content of Androgen Receptor in Cultured Genital Skin Fibroblast From Different Ages of Chinese Normal Men

    Institute of Scientific and Technical Information of China (English)

    卢建; 何立敏; 张金山; 杨震; 周云

    1995-01-01

    A ratpid, simple, reliable method is described for assaying androgen receptor (AR) in dispersed, whole, cultured human genital skin fibroblasts (GSF) with a synthetic androgen, 3H-methyltrienolone (3H-R1881). Receptors for androgen in GSF exhiblt high affinity (Kd=3.0±0.1 nmol/L), low binding capacity and androgen specificity. The content of AR in cultured GSF from 40 normal men varying in age from 1.5—60 years u:as also investigated by this assay. Scatchard analysis and slngle plot revealed the presence of 4.500-8500 binding sites per cell, mean number of AR in GSF of these men is 6288±1082 binding sites/cell. No significant difference was observed in the content of AR in different age groups. This result showed that the content of AR in these ceils did not change with age.

  7. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    Science.gov (United States)

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.

  8. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

    Science.gov (United States)

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

    Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

  9. Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species.

    Science.gov (United States)

    Mestre-Citrinovitz, Ana Cecilia; Sestelo, Adrián Jorge; Ceballos, María Belén; Barañao, José Lino; Saragüeta, Patricia

    2016-10-10

    Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar. The fibroblasts obtained are a part of the Genetic Bank of Buenos Aires Zoo with the 6700 samples, including tissues such as muscle, ovarian, testicular, blood, fibroblast cultures, sperm, hair, and fluids and cells from 450 individuals of 87 different species. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  10. Developing a culturally based cardiac rehabilitation program: the HELA study.

    Science.gov (United States)

    Look, Mele A; Kaholokula, Joseph Keawe; Carvhalo, Amy; Seto, Todd; de Silva, Mapuana

    2012-01-01

    Heart disease disproportionately affects Native Hawaiians and other Pacific people. In response, researchers proposed and communities endorsed, developing a cardiac rehabilitation (CR) program based on the hula, a Native Hawaiian dance form. The utilization of cultural practices in health interventions can improve outcomes and increase enrollment and retention, but requires sensitivity and understanding. This paper provides the conceptual framework and methods used for integration of multiple communities' perspectives to inform the design of a hula-based CR intervention. Specific strategies and processes were established to ensure the equity of scientific-clinical and patient- cultural knowledge and perspectives. Multiple methods were used and a flow diagram defined steps for the intervention development. Patient and cultural consultations provided information about the multidimensional benefits of hula and its use in a CR intervention. Clinical and scientific consultations provided specific guidelines for exercise prescription and patient monitoring. Integrating findings from all consultations identified important direction and requirements. Community-based participatory research (CBPR) principles guided a complex collaboration of multiple communities; although time consuming, inclusive consultations provided valuable information and relationships.

  11. In Vitro Wound Healing Improvement by Low-Level Laser Therapy Application in Cultured Gingival Fibroblasts

    Directory of Open Access Journals (Sweden)

    Fernanda G. Basso

    2012-01-01

    Full Text Available The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3×104 cells/cm2 were seeded on 24-well acrylic plates using plain DMEM supplemented with 10% fetal bovine serum. After 48-hour incubation with 5% CO2 at 37°C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780±3 nm; 40 mW with energy doses of 0.5, 1.5, 3, 5, and 7 J/cm2. Cells were irradiated every 24 h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3 J/cm2 were selected to evaluate the cell number (trypan blue assay and the cell migration capacity (wound healing assay; transwell migration assay. Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5%. Irradiation of the fibroblasts with 0.5 and 3 J/cm2 resulted in significant increase in cell metabolism compared with the nonrradiated group (P<0.05. Both energy doses promoted significant increase in the cell number as well as in cell migration (P<0.05. These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro.

  12. Bifunctional Effect of Human IFN-γ on Cultured Human Fibroblasts from Tenon‘s Capsule

    Institute of Scientific and Technical Information of China (English)

    YanGuo; JianGe; 等

    2002-01-01

    Purpose:To study the effect of human IFN-γ on in ivtro cultured human fibroblasts from Tenon's capsule.Materials and methods:The effect of different concentrations of human IFN-γand mitomycin-C (MMC),5-fluorouracil(5-Fu) on cultured human Tenon's capsule fibroblasts(HTCF) was measured using a MTT[3-(4,5-dimethylthiazo-2-yI)]-2,5-diphenylterazolium bromide;Thiazolyl blue) colorimetric assay.The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0 version.The difference was considered to be significant if P<0.05.Results:The effects of MMC and 5-Fu on the growth of HTCF were negative,while the effects of IFN-γon the growth of HTCF were both negative(102-104 units/ml in two experiments)and positive(106,105,10 units /ml in two experiments).The inhibition rate of MMC ranged from 5.73% to 46.9% ,which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92%(P=0.351).The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu (P<0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition)on proliferation of cultured HTCF.The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu.Further study has to be carried out to document theinhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation.Eye Science 2000;16:43-47.

  13. Bifunctional Effect of Human IFN-γon Cultured Human Fibroblasts from Tenon's Capsule

    Institute of Scientific and Technical Information of China (English)

    Yan Guo; Jian Ge; Haiquan Liu; Yanyan Li; Jianliang Zheng; Xiangkun Huang; Yuqing Lan

    2000-01-01

    Purpose: To study the effect of human IFN-γ on in vitro cultured human fibroblasts from Tenon's capsuleMaterials and methods: The effect of different concentrations of human IFN-γ and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MIT [3-(4, 5-dimethylthiazo-2-yl)] -2,5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0version. The difference was considered to be significant if P < 0. 05.Results: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-γ on the growth of HTCF were both negative (102 ~ l04 units/ml in two experiments) and positive (106, 105, 10 units/ml in two experiments) . The inhibition rate of MMC ranged from 5.73% to 46. 9%, which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92% ( P= 0. 351) . The inhibition rate of IFN-γ in two experiments was smaller than MMC and 5-Fu ( P < 0.05).Conclusion: IFN-γ has bifunctional effect (both enhancement and inhibition) on proliferation of cultured HTCF. The antiproliferative effect of IFN-γ was weaker than MMC and 5-Fu. Further study has to be carried out to document the inhibition of scar formation of filtration bleb by IFN-γ and the molecular mechanisms of its bifunctional effect on HTCF proliferation. Eye Science 2000; 16: 43~ 47.

  14. Effect of Qiangxin Decoction on Cardiac Fibroblast Proliferation and Collagen Synthesis%中药强心饮含药血清对心肌成纤维细胞增殖和胶原合成的影响

    Institute of Scientific and Technical Information of China (English)

    沈雁; 严世芸

    2011-01-01

    Objective: To investigate the effect of Qiangxin Decoction on the proliferation and collagen synthesis in neonatal rats cardiac fibroblast(CFs) induced by tumor necrceis factor α (TNF -α) ,and to explore the mechanism of Qiangxin Decoction on reversing cardiac fibrosis. Methods: Cardiac fibroblasts derived from neonatal rats were cultured with enzymatic dissociation, and CFs pathological model was induced by tumor necrosis factor at ( TNF - α). Thiazolyl blue MTI assay and hydroxyproline were employed to measure Cfs proliferation and collagen synthesis in order to determine the effect of different dosage decoction on CFs proliferation and collagen synthesis. Resu/ts :Compare with control group, TNF -α could induce cardiac fibroblast proliferation and increase collagen synthesis significantly( P < 0.05) ,wlule Qiangxin Decoction inhibit Cfs proliferation and collagen synthesis (P < 0. 05). Conclusion:Qiangxin Decoction could reverse cardiac fibrosis,which may be related to inhibit CFs proliferation and collage synthesis induced by TNF- α.%目的:研究强心饮对肿瘤坏死因子α(tumor necrosis factor α,TNF-α)诱导新生大鼠心肌成纤维细胞(cardi-ac fibroblasm CFs)增殖和胶原合成的影响,探讨其抑制心肌纤维化的作用机制.方法:采用胰酶消化法分离培养孔鼠CFs,建立TNF-α诱导新生大鼠CFs纤维化病理模型,分别采用四氮喳篮(MTT)比色法、羟脯氨酸法检测不同浓度强心饮含药血清对CFs增殖和胶原含量影响.结果:TNF-α能够刺激CFs增殖和胶原合成,与空白组相比差异显著(P<0.05);强心饮抑制TNF-α诱导CFs增殖和胶原合成(P均<0.05).结论:强心饮通过抑制TNF-α诱导的CFs增殖和胶原合成,发挥有效的抗心肌纤维化作用.

  15. Metalloproteinase 11, potential marker and molecular target in advanced and castration-resistant prostate cancer. Culture study of peritumoral fibroblasts.

    Science.gov (United States)

    Fernandez-Gomez, J M; Eiro, N; García-Rodríguez, J J; Quintás-Blanco, A; Gonzalez-Ruiz de León, C; Perez de Haro, M L; Vizoso-Piñero, F

    To analyze the expression of metalloprotein 11 (MMP11) in cultured fibroblasts obtained from human prostate tumors with different clinical and pathological characteristics. For this study we analyzed samples of transrectal prostate biopsies from tumors with different characteristics, treated with or whithout androgen deprivation (AD). After optimization of the culture method, fibroblasts were isolated and cultured to perform the study (PCR) of MMP11 mRNA. Finally, 37 cases were studied: 5 samples of benign prostatic hyperplasia, 14 cases with localized neoplasms (7 high-risk according to the D'Amico classification), 5 with metastasic tumors (bone metastases), and 13 treated with AD therapy, of which 6 fulfilled the requirements to be defined as resistant to castration. In tumors without AD therapy, MMP11 expression was significantly higher (P=.001) in fibroblasts of higher grade tumors. A significant (P=.001) correlation was found between PSA and expression of MMP11 in fibroblast s and a significant increase of MMP11 expression in metastatic tumors. In tumors with AD therapy, a significantly greater expression of MMP11 was observed in resistant to castration patients than in those sensitive to castration (P=.003). In advanced prostate tumors or in stages of increased tumor aggressiveness, the production of MMP11 by fibroblasts is significantly greater than in non-metastatic tumors or in AD sensitive tumors. Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  16. Fibroblasts weaken the anti-tumor effect of gefitinib on co-cultured non-small cell lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Xiao; Wang Peiqin; Jiang Tao; Yu Wenchen; Shang Yan; Han Yiping; Zhang Pingping

    2014-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common lung malignancy worldwide.The metastatic potential of NSCLC cells has been shown to be associated with the tumor microenvironment,which consists of tumor cells,stroma,blood vessels,immune infiltrates and the extracellular matrix.Fibroblasts can produce numerous extraceilular matrix molecules and growth factors.Gefitinib has been evaluated as a first-line treatment in selected patients,and it has shown favorable efficacy especially in NSCLC,but it is not effective for everyone.Methods In this study,we examined the antitumor activity of gefitinib on lung fibroblasts co-cultured of lung cancer cells.A series of co-culture experiments that employed cell counting kit-8 (CCK8),transwells,real-time polymerase chain reaction (RT-PCR) and Western blotting with HFL-1 fibroblasts and A549 human lung carcinoma cells were performed to learn more about tumor cell proliferation,migration and invasion; and to determine any change of epithelial mesenchymal transition (EMT)-associated tumor markers vimentin,matrix metallopro-teinase 2 (MMP2) and chemotaxis cytokines receptor 4 (CXCR4) mRNA levels.Results A549 cell proliferation in the presence of HFL-1 cells was not significantly increased compared with A549 cells alone,but A549 cell spheroid body formation was increased after co-culture,and treatment with gefitinib increased further.Our study also revealed that fibroblasts attenuated the lung cancer cell inhibition ratio of migration and invasion after gefitinib treatment in vitro.To further study this mechanism,RT-PCR analysis showed that vimentin,MMP2 and CXCR4 mRNA levels were more highly expressed in the lung cancer cells after co-culture,but did not obviously decrease compared with the control cells following gefitinib treatment.This suggests the mechanism by which fibroblasts attenuate gefitinib-induced expression of EMT-associated tumor markers.Finally,our results demonstrated that co-culture with A549 lung

  17. Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials.

    Science.gov (United States)

    Lombello, C B; Santos, A R; Malmonge, S M; Barbanti, S H; Wada, M L F; Duek, E A R

    2002-09-01

    Cell adhesion is influenced by the physical and chemical characteristics of the materials used as substrate for cell culturing. In this work, we evaluated the influence of the morphological and chemical characteristics of different polymeric substrates on the adhesion and morphology of fibroblastic cells. Cell growth on poly (L-lactic acid) [PLLA] membranes and poly(2-hydroxy ethyl methacrylate) [polyHEMA], poly(2-hydroxy ethyl methacrylate)-cellulose acetate [polyHEMA-CA] and poly(2-hydroxy ethyl methacrylate)-poly(methyl methacrylate-co-acrylic acid) [polyHEMA-poly(MMA-co-AA)] hydrogels of different densities and pore diameters was examined. Cells adhered preferentially to more negatively charged substrates, with polyHEMA hydrogels being more adhesive than the other substractes. The pores present in PLLA membranes did not interfere with adhesion, but the cells showed a distinctive morphology on each membrane.

  18. Expression of the familial Mediterranean fever gene and activity of the C5a inhibitor in human primary fibroblast cultures.

    Science.gov (United States)

    Matzner, Y; Abedat, S; Shapiro, E; Eisenberg, S; Bar-Gil-Shitrit, A; Stepensky, P; Calco, S; Azar, Y; Urieli-Shoval, S

    2000-07-15

    Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)-8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression of MEFV and C5a/IL-8-inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8-inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1 beta. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1 beta. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8-inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation. (Blood. 2000;96:727-731)

  19. Poly[tri(ethylene glycol) ethyl ether methacrylate]-coated surfaces for controlled fibroblasts culturing.

    Science.gov (United States)

    Dworak, Andrzej; Utrata-Wesołek, Alicja; Szweda, Dawid; Kowalczuk, Agnieszka; Trzebicka, Barbara; Anioł, Jacek; Sieroń, Aleksander L; Klama-Baryła, Agnieszka; Kawecki, Marek

    2013-03-01

    Well-defined thermosensitive poly[tri(ethylene glycol) monoethyl ether methacrylate] (P(TEGMA-EE)) brushes were synthesized on a solid substrate by the surface-initiated atom transfer radical polymerization of TEGMA-EE. The polymerization reaction was initiated by 2-bromo-2-methylpropionate groups immobilized on the surface of the wafers. The changes in the surface composition, morphology, philicity, and thickness that occurred at each step of wafer functionalization confirmed that all surface modification procedures were successful. Both the successful modification of the surface and bonding of the P(TEGMA-EE) layer were confirmed by X-ray photoelectron spectroscopy (XPS) measurements. The thickness of the obtained P(TEGMA-EE) layers increased with increasing polymerization time. The increase of environmental temperature above the cloud point temperature of P(TEGMA-EE) caused the changes of surface philicity. A simultaneous decrease in the polymer layer thickness confirmed the thermosensitive properties of these P(TEGMA-EE) layers. The thermosensitive polymer surfaces obtained were evaluated for the growth and harvesting of human fibroblasts (basic skin cells). At 37 °C, seeded cells adhered to and spread well onto the P(TEGMA-EE)-coated surfaces. A confluent cell sheet was formed within 24 h of cell culture. Lowering the temperature to an optimal value of 17.5 °C (below the cloud point temperature of the polymer, TCP, in cell culture medium) led to the separation of the fibroblast sheet from the polymer layer. These promising results indicate that the surfaces produced may successfully be used as substrate for engineering of skin tissue, especially for delivering cell sheets in the treatment of burns and slow-healing wounds.

  20. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Science.gov (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  1. Cardiac myocyte diversity and a fibroblast network in the junctional region of the zebrafish heart revealed by transmission and serial block-face scanning electron microscopy.

    Science.gov (United States)

    Lafontant, Pascal J; Behzad, Ali R; Brown, Evelyn; Landry, Paul; Hu, Norman; Burns, Alan R

    2013-01-01

    The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.

  2. Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

    KAUST Repository

    Lafontant, Pascal J.

    2013-08-23

    The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart. © 2013 Lafontant et al.

  3. Electrochemical properties of 316L stainless steel with culturing L929 fibroblasts.

    Science.gov (United States)

    Hiromoto, Sachiko; Hanawa, Takao

    2006-08-22

    Potentiodynamic polarization and impedance tests were carried out on 316L stainless steel with culturing murine fibroblast L929 cells to elucidate the corrosion behaviour of 316L steel with L929 cells and to understand the electrochemical interface between 316L steel and cells, respectively. Potential step test was carried out on 316L steel with type I collagen coating and culturing L929 cells to compare the effects of collagen and L929 cells. The open-circuit potential of 316L steel slightly shifted in a negative manner and passive current density increased with cells, indicating a decrease in the protective ability of passive oxide film. The pitting potential decreased with cells, indicating a decrease in the pitting corrosion resistance. In addition, a decrease in diffusivity at the interface was indicated from the decrease in the cathodic current density and the increase in the diffusion resistance parameter in the impedance test. The anodic peak current in the potential step test decreased with cells and collagen. Consequently, the corrosion resistance of 316L steel decreases with L929 cells. In addition, collagen coating would provide an environment for anodic reaction similar to that with culturing cells.

  4. Effect of salicyclic acid on gluccorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma.

    Science.gov (United States)

    Koshihara, Y; Yamagishi, M; Murota, S I

    1976-06-23

    The binding activity of [3H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [3H]dexamethasone of 3.64 - 10(-8) M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 - 10(5). Total binding activity to [3H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid was at 37 degrees C. The maximum enhancement was seen at the concentration of 10(-3)M and in 3h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicyclic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin,also enhanced the total binding activity to [3H]dexamethasone of the cytoplasmic fraction at the concentration of 2 - 10(-5) M and 2 - 10(-7) M, respectively.

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

    Science.gov (United States)

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

    2015-12-01

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

  6. The effect of exposure duration of self etch dentin bonding on the toxicity of human gingival fibroblast of cell culture

    Directory of Open Access Journals (Sweden)

    Sri Lestari

    2008-06-01

    Full Text Available Self etch dentin bonding created to make light easily activate the application of composite resin on tooth surface. The monomer content has acid effect that could irritate tooth pulp. The purpose of this study was to evaluate the effect of light exposure duration of self etch dentin bonding on toxicity of human gingival fibroblast of cell culture by MTT assay. Self etch dentin bonding was used as on experimental unit and the sample was exposed by visible light curing in different duration: 10, 20, 30 seconds and immerged in artificial saliva in pH 7 for 24 hours. 100 µl artificial saliva was exposed to human gingival fibroblast of cell culture 20.000 cells/100 µl RPMI for 24 hours. Toxicity was evaluated by MTT assay, optical density was measured using 550 nm spectrophotometer. The data was analyzed using Kruskal Wallis in 5% degree of significance. The result showed that increasing exposure duration (10, 20, 30 seconds of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture. It is concluded that 30 seconds-exposure of self etch dentin bonding will reduce the toxicity of human gingival fibroblast of cell culture.

  7. Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels.

    Science.gov (United States)

    Nagano, Takashi; Hao, Ji-Long; Nakamura, Masatsugu; Nishida, Teruo

    2002-08-01

    To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.

  8. Pengaruh Cairan Cultur Filtrate Fibroblast (CFF Terhadap Penyembuhan Luka; Penelitian eksperimental pada Rattus Norvegicus Galur Wistar

    Directory of Open Access Journals (Sweden)

    Oky Masir

    2012-11-01

    Full Text Available AbstrakLatar belakang:Metode penyembuhan luka telah mengalami perkembangan, baik berupa suatu produk atau stimulan terhadap proses biologis tubuh dalam menkompensasi luka. Fibroblas merupakan salah satu komponen penyembuhan yang berperan penting dalam proses fibroplasia. Culture Filtrate Fibroblast (CFF merupakan hasil kultur fibroblas yang akan dibuktikan efeknya terhadap proses percepatan penyembuhan luka pada penelitian ini. Metode. Penelitian ini menggunakan desain eksperimental dengan metode post test only control group design dan rancangan acak kelompok (RAK dengan menggunakan tikus putih wistar. Hewan coba dibagi menjadi 4 kelompok, yaitu 2 kelompok perlakuan yang diberikan CFF ke area eksisi luka dan kelompok kontrol yang diberikan larutan NaCl 0,9% ke area eksisi luka. Data diolah dengan SPSS 16.0. Data Kategori dianalisa dengan Chi-squared dan data numerik dengan Independent T-test. Hasil. Dari tingkat penyembuhan tidak ditemukan perbedaan pada kedua kelompok, namun perubahan restriksi jaringan lebih besar pada kelompok perlakuan. Pada skor pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru pada hari ke-3 tidak ditemukan perbedaan antara kedua kelompok. Namun pada pengamatan hari ke-7 memperlihatkan pembentukan kolagen, derajat epitelisasi serta jumlah pembentukan pembuluh darah baru lebih banyak pada kelompok perlakuan. Pada fibrosis hari ke-3 dan hari ke-7 memperlihatkan terjadinya fibrosis lebih banyak pada kelompok perlakuan dibanding kontrol. Pada pengamatan terjadinya infeksi hari ke-3 memperlihatkan infeksi lebih sedikit pada kelompok perlakuan dan terjadinya infeksi sama pada hari ke-7. Kesimpulan. CFF memberikan tingkat penyembuhan luka yang lebih baik dibanding NaCl.Kata kunci: CFF, NaCl 0,9 %, tingkat penyembuhan luka.Abstract Background: Wound healing methods have been developed, either a product or a stimulant to the body's biological processes in wound compensation. Fibroblasts is one

  9. CULTURES OF FIBROBLAST-LIKE SYNOVIAL CELLS FROM PATIENTS WITH RHEUMATOID ARTHRITIS: PROPERTIES AND OPPORTUNITIES

    Directory of Open Access Journals (Sweden)

    M. A. Schneider

    2016-01-01

    Full Text Available The present review contains data from literature concerning the in vivo structure of synovial membranes in healthy people and patients with rheumatoid arthritis (RA. The properties of in vitro cultured fibroblast-like synovial cells (FLS from RA patients are considered, including FLS morphology, phenotype and function. A standard protocol of in vitro FLS culturing is described. Notably, the FLS are characterized by autonomic functioning, ability for invasive growth/migration, e.g., into non-affected joints. These FLS properties may a reason of multiple joint involvement typical to RA. Special attention is drawn to characterization of stable phenotypic profile of FLS which results from certain epigenetic disturbances, i.e., changes of the DNA methylation, histone acetylation, and micro-RNA effects.The FLS from RA patients are characterized with stable and extensive hypomethylation of genes which occurs in vivo and persists after repeated culture passages. Some promoters of genes involved into RA pathogenesis (for example, CXCL12, IL-6 are hypomethylated. By contrary, some other gene promoters (e.g., the death receptor 3 gene are shown to be hypermethylated. An increased histone acetylation of genes encoding proinflammatory mediators (such as MMP1 may be an important mechanism of persistent inflammation in RA. Changes in histone acetylation in FLS are related to high levels of ubiquitin-like SUMO-1 protein and concurrent decrease in specific protease SENP1activity. A role of histone acetylation in RA pathogenesis is supported by efficacy of a histone deacetylase inhibitor (Trichostatin A in collagen-induced murine arthritis. Local concentrations of micro RNA-155, micro-RNA-146а, and micro-RNA-203 are permanently increased in FLS cultures, synovial tissues, and PBMC of the RA patients. Expression of micro RNA-124а is decreased in FLS from RA, as compared with OA FLS.One may conclude that the fibroblast-like synovial cells are key cellular

  10. Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients

    Directory of Open Access Journals (Sweden)

    B. Villaggio

    2012-12-01

    Full Text Available Endothelin-1 (ET-1 seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxentan and ETA/BRA-bosentan on type I collagen (COL-1, fibronectin (FN and fibrillin-1 (FBL-1 synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM for 24 and 48 hrs with or without pre-treatment (1 hr with ETARA (2 μM or ETA/BRA (10 μM. Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

  11. Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

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    Lucie Germain

    2013-02-01

    Full Text Available A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3 can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

  12. Lunasin Inhibits Cell Proliferation via Apoptosis and Reduces the Production of Proinflammatory Cytokines in Cultured Rheumatoid Arthritis Synovial Fibroblasts

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    Shaohui Jia

    2015-01-01

    Full Text Available Lunasin, a peptide with 43 amino acid residues and initially isolated and identified in soybean cotyledon, has gained extensive attention due to its anti-inflammatory and anticancer properties. However, its treatment efficacy on rheumatoid arthritis (RA and corresponding mechanisms have not been reported. Herein, the synovial fibroblasts harvested and isolated from patients with RA were treated with lunasin at various concentrations to examine the proliferation, apoptosis status, and corresponding cell cycle of cultured RA synovial fibroblasts. Meanwhile, the underlying mechanisms of lunasin for RA treatment are explored through Western blot, real-time PCR, ELISA, and luciferase reporter assays. Lunasin significantly inhibited the proliferation and induced the apoptosis of cultured RA synovial fibroblasts. In addition, lunasin reduced the production of interleukin-6 (IL-6, IL-8, and matrix metalloproteinase-3 (MMP-3 and suppressed the activation of NF-κB in cultured RA synovial fibroblasts but did not reveal obvious modulation on the secretion and gene expression of MMP-1. Therefore, lunasin will have promising potential as a novel nutritional supplement or drug candidate for RA due to its potency of suppressing synovial cell proliferation and decreasing the production of proinflammatory cytokines and MMPs in synovial cells.

  13. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.

    Science.gov (United States)

    Pfefferkorn, E R; Guyre, P M

    1984-01-01

    The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii. Images PMID:6425215

  14. Curvature-Dependent Excitation Propagation in Cultured Cardiac Tissue.

    Science.gov (United States)

    Kadota, S; Kay, M W; Magome, N; Agladze, K

    2012-02-01

    The geometry of excitation wave front may play an important role on the propagation block and spiral wave formation. The wave front which is bent over the critical value due to interaction with the obstacles may partially cease to propagate and appearing wave breaks evolve into rotating waves or reentry. This scenario may explain how reentry spontaneously originates in a heart. We studied highly curved excitation wave fronts in the cardiac tissue culture and found that in the conditions of normal, non-inhibited excitability the curvature effects do not play essential role in the propagation. Neither narrow isthmuses nor sharp corners of the obstacles, being classical objects for production of extremely curved wave front, affect non-inhibited wave propagation. The curvature-related phenomena of the propagation block and wave detachment from the obstacle boundary were observed only after partial suppression of the sodium channels with Lidocaine. Computer simulations confirmed the experimental observations. The explanation of the observed phenomena refers to the fact that the heart tissue is made of finite size cells so that curvature radii smaller than the cardiomyocyte size loses sense, and in non-inhibited tissue the single cell is capable to transmit excitation to its neighbors.

  15. Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, Marta; Hamilton, Tiffani; Haili Li [Utah Univ., Salt Lake City, UT (United States). Dept. of Internal Medicine

    1995-09-01

    The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author).

  16. AGEs and Glucose Levels Modulate Type I and III Procollagen mRNA Synthesis in Dermal Fibroblasts Cells Culture

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    Serban Iren Andreea

    2008-01-01

    Full Text Available In the dermis, fibroblasts play an important role in the turnover of the dermal extracellular matrix. Collagen I and III, the most important dermal proteins of the extracellular matrix, are progressively altered during ageing and diabetes. For mimicking diabetic conditions, the cultured human dermal fibroblasts were incubated with increasing amounts of AGE-modified BSA and D-glucose for 24 hours. The expression of procollagen α2(I and procollagen α1(III mRNA was analyzed by quantitative real-time PCR. Our data revealed that the treatment of fibroblasts with AGE-modified BSA upregulated the expression of procollagen α2(I and procollagen α1(III mRNA in a dose-dependent manner. High glucose levels mildly induced a profibrogenic pattern, increasing the procollagen α2(I mRNA expression whereas there was a downregulation tendency of procollagen α1(III mRNA.

  17. Aislamiento y cultivo de fibroblastos endoneurales Endoneural fibroblasts isolation and culture

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    Spinel Clara

    2004-12-01

    time remodel tissues that are being repaired. Dermal fibroblasts are well studied both in vitro and in vivo, and are used to regenerate dermal EMC which in turn supports the regeneration of the epidermis. Confluence of dermal or periodontal fibroblasts takes place between 8 and 10 days of culture. In the process of regeneration of damaged peripheral nerves, Schwann's cells secrete neurotrophic and neurotropic growth factors and some of the EMC elements needed for regeneration to take place, which makes them the most studied and used cells in culture. So far, endoneural fibroblasts (EF have not been considered as important elements in nerve regeneration, mainly because they may occasionally form fibromes that hinder regeneration. But there is evidence that they may play a role in the remodeling of the EMC, through the secretion of metalloproteins that modify the pre-Nerve Growth Factor (preNGF secreted by Schwann's cells into active NGF, which promotes neurites regeneration. The aim of this study was is to isolate EF from sciatic nerves taken from mature rats, and to obtain them in purified culture. A number of methods of dissection and digestion were developed to obtain primary pure EF cultures as well as to study them in the way Schwann's cells have been studied. Selective isolation of EF was accomplished, reaching confluence between the fourth and the fifth day in monolayer primary culture. Producing a population of EF will make it possible to carry out studies in tridimensional culture and in prosthesis in order to define and develop new alternatives for the regeneration of peripheral nerves.

  18. Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells.

    Science.gov (United States)

    Ishii, Kenichiro; Mizokami, Atsushi; Tsunoda, Toshiyuki; Iguchi, Kazuhiro; Kato, Manabu; Hori, Yasuhide; Arima, Kiminobu; Namiki, Mikio; Sugimura, Yoshiki

    2011-12-01

    In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.

  19. Stochastic Alternating Dynamics for Synchronous EAD-Like Beating Rhythms in Cultured Cardiac Myocytes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ning; ZHANG Hui-Min; LIU Zhi-Qiang; DING Xue-Li; YANG Ming-Hao; GU Hua-Guang; REN Wei

    2009-01-01

    Dissolved cardiac myocytes can couple together and generate synchronous beatings in culture. We observed a synchronized early after-depolarization(EAD)-like rhythm in cultured cardiac myocytes and reproduced the experimental observation in a network mathematical model whose dynamics are close to a Hopf bifurcation. The mechanism for this EAD-like rhythm is attributed to noised-induced stochastic alternatings between the focus and the limit cycle. These results provide novel understandings for pathological heart rhythms like the early immature beatings.

  20. Influence of the fatty acid composition of high-density lipoprotein phospholipids on the cholesterol efflux from cultured fibroblasts.

    Science.gov (United States)

    Esteva, O; Baudet, M F; Lasserre, M; Jacotot, B

    1986-02-12

    The purpose of this work was to determine whether the changes induced by dietary manipulations in the chemical composition of high-density lipoproteins (HDL) (particularly phospholipid fatty acid composition) modified their capacity to promote [3H]cholesterol efflux from cultured fibroblasts. Plasma HDL were obtained from subjects fed for six successive long periods on diets consisting of one predominant fat: peanut oil, corn oil, olive oil, soybean oil, low erucic acid rapeseed oil or milk fats. The [3H]cholesterol efflux from cells in the presence of plasma HDL was studied by means of normal adult human fibroblasts in culture. The [3H]cholesterol efflux from fibroblasts appeared to be independent of the overall composition of HDL and of the degree of saturation of the HDL phospholipid fatty acids, but it was correlated with the phospholipid fatty acid chain length. The [3H]cholesterol efflux from fibroblasts is highly and positively correlated with the sum of the HDL phospholipid C20, C22, C24 fatty acids, and negatively correlated with the sum of the HDL phospholipid C18 fatty acids.

  1. Effects of bosentan on collagen type I synthesis on in vitro culture of scleroderma skin fibroblasts

    Directory of Open Access Journals (Sweden)

    S. Soldano

    2011-01-01

    Full Text Available The present study evaluated the effects of a non-selective endothelin (ETA/B receptors antagonist, on collagen type I (COLI synthesis on in vitro culture of scleroderma (SSc skin fibroblasts (Fb. Fb were obtained from skin biopsies of 6 female SSc patients (mean age 64. 1±6 years, after informed consent and Ethical Committee Approval. Cells were treated with endothelin-I [ET-I, 100nM] for 24 and 48 hrs, pre-treated for I hr with ETA/B receptors antagonist [10nM] alone or followed by ET-I for 24 and 48 hrs. Untreated Fb were used as controls. Immunocytochemistry and western blot analysis were performed to evaluate COLI synthesis. ET-I increased COLI synthesis both at 24 and 48 hrs when compared to controls. ETA/B receptor antagonost blocks the increased COLI synthesis ET-I-mediated both at 24 and 48 hrs vs. ET-I. Results showed that ET-I receptors blockage by ETA/B receptors antagonist might prevent the excessive synthesis of COLI, supporting its positive action in the management of skin fibrosis.

  2. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    Science.gov (United States)

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

  3. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

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    Laura N Bonifacio

    Full Text Available Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  4. Cultured skin keratinocytes from both normal individuals and basal cell naevus syndrome patients are more resistant to. gamma. -rays and UV light compared with cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, M.; Thacker, S.; Taylor, A.M.R. (Birmingham Univ. (UK). Cancer Research Campaign Labs.)

    1989-07-01

    Measurement of colony-forming ability following exposure to {gamma}-rays was performed on cultured skin keratinocytes and skin fibroblasts from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. Survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to {gamma}-rays or 254 nm UV light. An increased D{sub 37} rather than an increased D{sub o} was therefore the feature of such curves, contrasting with SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased D{sub o}. No difference in survival was observed between BCNS and normal cells following exposure to either UV or {gamma}-rays. (author).

  5. Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three-dimensional magnetic cell culture array.

    Science.gov (United States)

    Okochi, Mina; Matsumura, Taku; Yamamoto, And Shuhei; Nakayama, Eiichi; Jimbow, Kowichi; Honda, Hiroyuki

    2013-01-01

    A three-dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force-based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array-like multicellular patterns of melanoma were developed using a pin-holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct-interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect-interaction model) of melanoma spheroids, and (iii) fibroblast-sheets coexisting under melanoma spheroids (fibroblast-sheet model). The fibroblast-sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast-sheet model, the expression of IL-8 and MMP-2 increased by 24-fold and 2-fold, respectively, in real time RT-PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment.

  6. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    Science.gov (United States)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  7. In vitro clonogenic capacity of periodontal ligament fibroblasts cultured with Emdogain.

    Science.gov (United States)

    Ashkenazi, Malka; Shaked, Ilanit

    2006-02-01

    The aim of the present study was to evaluate the efficiency of Emdogain (EMD) in preserving the size of the periodontal ligament progenitor pool (clonogenic capacity) and in promoting their proliferation. Periodontal ligament fibroblasts (PDLF) were obtained from explants of young permanent healthy tooth. After initial outgrowth (10 days to 2 weeks following explantation), the culture medium of experimental flasks was replaced with medium supplemented with 100 microg ml(-1) EMD, whereas the other served as controls and were fed with regular medium. Following 5 weeks, the cells were washed (3x), harvested (trypsin + EDTA), and evaluated for their viability. Viable cells from each group were inoculated into six 96-well plates at a concentration of one viable cell per two wells and were allowed to grow for 5 weeks. The percentage of cells with clonogenic capacity was determined as the number of colonies formed/number of cells seeded x 100 in the experimental and control groups. Three degrees of dish area coverage were utilized: up to 25%, between 25% and 75% and higher than 75%. This experiment was repeated four times from four different donors. A total of 2328 cells were evaluated, half of which, were cultured with EMD. The mean percentage of cells (from all donors) who exhibited any clonogenic capacity in the presence of EMD was comparable with that of cells cultured in the absence of EMD: 26.6 +/- 14.3% when compared with 34.6 +/- 20.6% respectively (P = 0.186). Similarly, the percentage of clones that proliferated to cover up to 25% of the well area was comparable in the two groups 7.5 +/- 8.6 for EMD-treated clones and 7.1 +/- 7.8 for untreated clones (P = 0.674). The percentage of clones that proliferated to cover 25% up to 75% of the well area was greater EMD-treated clones as compared with the untreated cells: 8.1 +/- 6.7% vs 3.8 +/- 3%. However this difference was not statistically significant (P = 0.277). In contrast, the percentage of clones that covered

  8. Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

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    Kyung Eun Sung

    Full Text Available The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com. We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

  9. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  10. INFLUENCE OF QUERCETIN AND X-RAY ON COLLAGEN SYNTHESIS OF CULTURED HUMAN KELOID-DERIVED FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    Xiao Long; Xuan Zeng; Fu-quan Zhang; Xiao-jun Wang

    2006-01-01

    Objective To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen Ⅰ and Ⅲ expression. mRNA expression of collagen Ⅰ and Ⅲ, and transforming growth factor (TGF)-β1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR.Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen Ⅰ and Ⅲ were down-regulated by quercetin and X-ray ( P<0.05), particularly collagen Ⅰ (P<0.05). mRNA expression of both collagen Ⅰ and Ⅲ in quercetin groups significantly decreased compared with that in control group (P<0.05 ), especially in the group treated with both quercetin and X-ray( P<0.01). mRNA level of TGF-β1 gene was down-regulated by quercertin (P<0.05).Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.

  11. The effect of activated alveolar macrophages on experimental lung emphysema development. II. The study of fibroblast and alveolar macrophage co-culture.

    Science.gov (United States)

    Sulkowska, M; Wołczyński, S; Sulkowski, S; Sobaniec-Lotowska, M; Chyczewski, L; Sulik, M; Kulikowski, M; Dziecioł, J; Berger, W

    1995-01-01

    The cell-cell interaction between fibroblasts and alveolar macrophages was examined using a co-culture system. Alveolar macrophages (AM) were harvested from the bronchoalveolar lavages (BAL) of rats with papain induced lung emphysema. The BCG-vaccine was applied as a macrophage mobilizing and activating agent. The morphological examinations carried out in scanning electron microscope (SEM) as well as the evaluation of the uptake of 3H-thymidine did not show any significant differences between respective co-cultures of fibroblasts and AM isolated both from the lungs of control and experimental animals (treated with BCG or papain, and BCG+papain). However, significant growth were noted in 3H-thymidine uptake between fibroblast cultures done with or without cells isolated from the lungs. The results obtained suggest that AM can promote fibroblast proliferation during the progression of experimental lung emphysema.

  12. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    Energy Technology Data Exchange (ETDEWEB)

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

  13. Human ovarian neoplasm cell CD147 stimulates production and activation of matrix metalloproteinases in co-cultures with mouse fibroblasts

    Institute of Scientific and Technical Information of China (English)

    YANG Hong; ZOU Wei; XIN Xiao-yan

    2005-01-01

    Objective: To investigate the expression of CD147 on human ovarian neoplasm cell lines and its influence on production and activation of matrix metallproteinases(MMPs). Methods: The expression of CD147 on different human ovarian neoplasm cell lines was studied by western blotting. Co-culture was carried out to investigate the stimulative effect of the positive expression CD147 cell HO-8910 on the production of MMPs of fibroblast cell in vitro. Zymography and immune blotting were used to study the production and activity of positive MMPs, at the time, to explore the relation between CD147 and MMPs. Results: CD147 was positively presented in 2 ovarian neoplasm cell lines(HO-8910,3-AO), but in SKOV3, TC-1,NIN3T3 cell was negative. MMP-2 and MMP-9 were detected by HO-8910 cell line, mouse fibroblast cell and co-culture cells; but the expression in co-culture cell is obviously higher than individual cultures of each type alone.CD147 stimulated MMPs in dose-dependent manner. Conclusion: CD147 causes increased production and activation of MMP-2, MMP-9.CD147 is probably a indirect marker of some ovarian cancer cells with invasion and metastasis.

  14. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

    Directory of Open Access Journals (Sweden)

    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  15. Dynamic behavior of fragile X full mutations in cultured female fetal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Yu-jie SUN; Xiao HAN

    2004-01-01

    AIM: To assess mitotic stability of the fragile X full mutations and its relationship with DNA methylation. METHODS:The length change of the expanded CGG repeats was examined and correlated it with the methylation status in the DNA samples isolated from the fibroblasts derived from a fragile X female fetus and a fragile X male adult, respectively.RESULTS: A dramatic instability of the expanded CGG repeats in the female fetal fibroblasts was observed. Southem blot analysis revealed that the 6.9-kb major expanded band detected in passage 2 was completely replaced by a 7.7-kb band after passage 30. Fibroblast clones derived from the passage 3 displayed an unstable expansion of the CGG repeat during clonal proliferation, while methylation status of the CGG repeat region was maintained. In contrast, in fragile X male fibroblasts the expanded CGG repeats were stable during clonal proliferation.CONCLUSION: The mitotic instability of expanded CGG repeat is not always restricted in early development window as proposed previously and other elements rather than DNA methylation could affect the stability of the expanded CGG repeats in fragile X female fetal fibroblast cells.

  16. 心脏成纤维细胞及其参与心房颤动发生的研究进展%Research on Cardiac Fibroblasts and Their Participation in Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    郭金蕾

    2013-01-01

    心脏成纤维细胞在心脏的病理生理方面都扮演着重要角色.从疾病的角度来看,来自心脏成纤维细胞的细胞外基质的增多所导致的心房纤维化对于心房颤动的发生与发展起着重要作用.过量的细胞外基质可阻断心肌束的连续性,进而导致局部传导障碍和折返性心律失常.同时,心脏成纤维细胞上表达的多种离子通道使得成纤维细胞还可以与心肌细胞耦联并影响其电生理特性.因此,进一步了解成纤维细胞及其在心房颤动中的参与作用有助于人们深入挖掘与认识抗心律失常的治疗靶点.%Cardiac fibroblasts play a crucial role in cardiac pathophysiology . In disease states, increased quantities of extracellular matrix produced by cardiac fibroblasts leads to cardiac fibrosis , which is known to play a key role in atrial fibrillation. Excessive extracellular matrix protein can interrupt cardiomyocyte bundles' continuity, resulting in local conduction disturbances and reentrant arrhythmias . Meanwhile , the cardiac fibroblasts express varieties of ion channels , which make fibroblasts coupling to cardiomyocytes and affecting their cellular electrical properties. Therefore, a better understanding of cardiac fibroblasts and their participation in atrial fibrillation will enhance our com - prehension of the targets for anti-arrhythmias.

  17. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    Science.gov (United States)

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  18. Strategies to Study Desmin in Cardiac Muscle and Culture Systems.

    Science.gov (United States)

    Diokmetzidou, Antigoni; Tsikitis, Mary; Nikouli, Sofia; Kloukina, Ismini; Tsoupri, Elsa; Papathanasiou, Stamatis; Psarras, Stelios; Mavroidis, Manolis; Capetanaki, Yassemi

    2016-01-01

    Intermediate filament (IF) cytoskeleton comprises the fine-tuning cellular machinery regulating critical homeostatic mechanisms. In skeletal and cardiac muscle, deficiency or disturbance of the IF network leads to severe pathology, particularly in the latter. The three-dimensional scaffold of the muscle-specific IF protein desmin interconnects key features of the cardiac muscle cells, including the Z-disks, intercalated disks, plasma membrane, nucleus, mitochondria, lysosomes, and potentially sarcoplasmic reticulum. This is crucial for the highly organized striated muscle, in which effective energy production and transmission as well as mechanochemical signaling are tightly coordinated among the organelles and the contractile apparatus. The role of desmin and desmin-associated proteins in the biogenesis, trafficking, and organelle function, as well as the development, differentiation, and survival of the cardiac muscle begins to be enlightened, but the precise mechanisms remain elusive. We propose a set of experimental tools that can be used, in vivo and in vitro, to unravel crucial new pathways by which the IF cytoskeleton facilitates proper organelle function, homeostasis, and cytoprotection and further understand how its disturbance and deficiency lead to disease.

  19. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  20. Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model

    Energy Technology Data Exchange (ETDEWEB)

    Horie, Masafumi [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Saito, Akira, E-mail: asaitou-tky@umin.ac.jp [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Mikami, Yu [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Ohshima, Mitsuhiro [Department of Biochemistry, Ohu University School of Pharmaceutical Sciences (Japan); Morishita, Yasuyuki [Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo (Japan); Nakajima, Jun [Department of Thoracic Surgery, Graduate School of Medicine, University of Tokyo (Japan); Kohyama, Tadashi; Nagase, Takahide [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We established three patient-paired sets of CAFs and NFs. Black-Right-Pointing-Pointer CAFs and NFs were analyzed using three-dimensional co-culture experiments. Black-Right-Pointing-Pointer CAFs clearly enhanced collagen gel contraction. Black-Right-Pointing-Pointer CAFs showed higher {alpha}-SMA expression than NFs. Black-Right-Pointing-Pointer CAFs were implicated in invasion and differentiation of lung cancer cells. -- Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher {alpha}-smooth muscle actin ({alpha}-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

  1. Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture.

    OpenAIRE

    Alfa, M J; DeGagne, P; Totten, P A

    1996-01-01

    Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, produces several factors that damage human cells. We used isogenic mutants of H. ducreyi 35000 to demonstrate that the hemolytic activity and the cytotoxic effect of H. ducreyi on human foreskin fibroblasts are due to the same toxin.

  2. Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy.

    Directory of Open Access Journals (Sweden)

    Raghav K Chhetri

    Full Text Available The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A or pre-malignant (MCF10DCIS.com MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF. This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios [1] were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled in vitro, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.

  3. The use of a biostatic fascia lata thigh allograft as a scaffold for autologous human culture of fibroblasts--An in vitro study.

    Science.gov (United States)

    Żurek, Jarek; Dominiak, Marzena; Botzenhart, Ute; Bednarz, Wojciech

    2015-05-01

    The method for covering gingival recession defects and augmenting keratinized gingiva involves the use of autogenuous connective tissue grafts obtained from palatal mucosa in combination with various techniques of flap repositioning or tunnel techniques. In the case of multiple gingival recession defects the amount of connective tissue available for grafting is insufficient. Therefore, the use of substitutes is necessary. The most widely used material in recent years has been the acellular dermal matrix allograft. The disadvantage of its application lies in the absence of cells and blood vessels, which increases incorporation time. Primary cultured human autologic fibroblasts are commonly used to optimize the healing process. The aim of this study was to examine the in vitro biocompatibility of human fascia lata allograft as a new scaffold for primary cultured human autologic fibroblasts. For that, a fibroblast culture obtained from a fragment of gingival tissue taken from the hard palate mucosa of a subject was used. After 14 days the colony cells were inoculated on a fragment of human fascia lata allograft. After a further 7 days of incubation the material was frozen, cut and prepared for histochemical examination. After two weeks of incubation, and 7 days after inoculation on a fragment of fascia lata allograft numerous accumulations of the cultured fibroblast were found that had a typical structure and produced collagen fibres. A human fascia lata allograft can be used as a scaffold for primary cultured human autologic fibroblasts. Further studies should confirm the clinical efficacy of this solution.

  4. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    Science.gov (United States)

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  5. Treatment of Hypertrophic Scar in Human with Autologous Transplantation of Cultured Keratinocytes and Fibroblasts along with Fibrin Glue

    Directory of Open Access Journals (Sweden)

    Ehsan Taghiabadi

    2015-04-01

    Full Text Available Objective: Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can’t be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue (FG was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. Materials and Methods: In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin (H&E and Masson’s Trichrome (MT staining of the biopsies after 8 weeks. Results: Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. Conclusion: These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate.

  6. Preoperative screening cultures in the identification of staphylococci causing wound and valvular infections in cardiac surgery

    NARCIS (Netherlands)

    E. J. Ridgway; A.P.R. Wilson; M. C. Kelsey

    1990-01-01

    textabstractCultures of nasal or presternal swabs form part of the routine preoperative screening of patients on the cardiac surgical ward. During a trial of antibiotic prophylaxis in 314 patients, preoperative isolates of Staphylococcus aureus and coagulase-negative staphylococci were compared with

  7. The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Eskildsen, Tilde V; Schneider, Mikael; Sandberg, Maria B;

    2015-01-01

    , signalling molecules and transcription factors. Subsequent comprehensive in silico analysis identified 24 target genes, of which 22 genes were qPCR validated. We identified seven genes involved in AngII signalling pathways. CONCLUSION: We here report novel insight of an extensive network of molecular......INTRODUCTION: MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular...... function including regulation of cardiac hypertrophy, heart failure and blood pressure possibly through AT1R signalling. However, the miR-132/212 targets in the heart remain unknown. MATERIALS AND METHODS: To understand the role of these miRNAs in cardiac signalling networks, we undertook comprehensive...

  8. Determination of cardiac glycosides and total phenols in different generations of Securigera securidaca suspension culture

    Directory of Open Access Journals (Sweden)

    Z. Tofighi

    2016-04-01

    Full Text Available Background and objectives: The seeds of Securigera securidaca (L. Deg. & Dorf. (Fabaceae are used as anti-diabetic remedy in Iranian folk medicine. The aim of the present study was to establish the callus and suspension culture of S. securidaca seeds for the first time and to determine the major secondary metabolites including cardiac glycosides and total phenols. Methods: The culture of S. securidaca from seeds was initiated in hormone-supplemented MS medium containing 1 and 0.1 ppm 2, 4-D solution for solid and suspension cultures, respectively, sucrose and vitamins (B1, B2, B6, Folic acid, Biotin, Nicotinamide and Ca pantothenate at 25 °C and 12 h photoperiods. The cardiac glycosides were determined based on the calibration curve of securidaside which was isolated from the seeds extract of S. securidaca. Total phenolic compounds of different generations of suspension culture were determined using Folin Ciocalteu reagent. Results: Callus culture of S. securidaca was grown light cream to pale yellow in color and soft in texture while the cells of suspension culture grew cream to yellow with isolated cells and small aggregates. The production of cardiac glycosides in the 7th generation were more than the seeds extract (p

  9. Genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast BHK-21 cell cultures

    Directory of Open Access Journals (Sweden)

    Ceples Dian Kartika W.P

    2015-03-01

    Full Text Available Background: Health industry has always used natural products as an alternative. Propolis, a natural antibiotic, is a resinous yellow brown or dark brown substance derived from honey bees (Apis mellifera. The main chemical compounds contained in propolis are flavonoids, phenolics and other various aromatic compounds. Flavonoids are well known plant compounds that have antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory proprieties. Propolis is expected to be an alternative used for root canal treatment with lower toxicity compared to calcium hydroxide (Ca(OH2 . Over the last decade, a new material, mineral trioxide aggregate (MTA was developed, and has been used as the gold standard. All materials used in mouth should be biocompatible. The initial level of material biocompatibility evaluation involves toxicity and genotoxicity tests. Purpose: This research is aimed to conduct comparison test of genotoxicity effect of propolis extract, MTA and Ca(OH2 on fibroblast BHK-21 cell culture. Methods: This research was conducted with single-cell gel electrophoresis method. Results: The results indicate that propolis extract cannot cause DNA damage, while MTA can cause apoptosis and Ca(OH2 can cause neucrosis. Conclusion: It can be concluded that propolis extract has genotoxicity effect lower than MTA and Ca(OH2 , but MTA has lower effect on fibroblast BHK-21 cell culture.

  10. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

    Science.gov (United States)

    Jubin, K; Martin, Y; Lawrence-Watt, D J; Sharpe, J R

    2011-12-01

    Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

  11. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

  12. Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

    2016-07-08

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

  13. The Cultural Meaning of Cardiac Illness and Self-Care Among Lebanese Patients With Coronary Artery Disease.

    Science.gov (United States)

    Dumit, Nuhad Yazbik; Magilvy, Joan Kathy; Afifi, Rima

    2016-07-01

    Cardiac disease is the leading cause of death in Lebanon, accounting for 22% to 26% of total deaths in the country. A thorough understanding of perceptions of cardiac illness and related self-care management is critical to the development of secondary prevention programs that are specific to the Lebanese culture. To explore the cultural perceptions of cardiac illness and the associated meaning of self-care among Lebanese patients. Using a qualitative descriptive method, semistructured interviews were conducted with a purposive sample of 15 Lebanese cardiac patients recruited from a medical center in Beirut, Lebanon. The qualitative descriptive analysis yielded one overarching and two other themes describing perceptions of cardiac illness and self-care within the Lebanese cultural context. The overarching cultural theme was, "Lebanese cardiac patients were unfamiliar with the term concept and meaning of self-care." Lebanese cardiac patients thanked God and accepted their fate (Theme I). The participants considered their cardiac incident a life or death warning (Theme II). Health care providers need to consider patients' cultural perception of illness while planning and evaluating cardiac self-care programs. © The Author(s) 2015.

  14. Transformation of adult rat cardiac myocytes in primary culture.

    Science.gov (United States)

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  15. Comparative effects of posterior eye cup tissues from myopic and hyperopic chick eyes on cultured scleral fibroblasts.

    Science.gov (United States)

    Christian, Parul G; Harkin, Damien G; Rayner, Cassie; Schmid, Katrina L

    2013-02-01

    The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5-8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA

  16. Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

    Energy Technology Data Exchange (ETDEWEB)

    Usuki, S.; Lyu, S.C.; Sweeley, C.C.

    1988-05-15

    Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ((1-14C)N-acetylmannosamine) and a radioactive precursor of ceramide ((3,3-3H2)serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.

  17. Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Crimi Nunzio

    2006-09-01

    Full Text Available Abstract Rationale Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition. Methods We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC in 5 primary lung fibroblast lines derived from normal subjects (NF and from 3 histologically proven IPF patients (FF. Results Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF. Conclusion These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

  18. Uroporphyria development in cultured chick embryo fibroblasts long-term treated with chloramphenicol and ethidium bromide.

    Science.gov (United States)

    de Muys, J M; Morais, R

    1984-07-23

    Long-term chloramphenicol- and ethidium bromide-treated chick embryo fibroblasts synthesize large amounts of porphyrins from exogenously added delta-aminolevulinic acid. The porphyrins consist mainly of uro- and heptacarboxyporphyrins and are retained within cells. Uroporphyria development is a time-dependent process which accompanies a step-wise decrease in the capacity of the mitochondrial respiratory chain. Upon removal of chloramphenicol from the medium, the pattern of porphyrin production readily returns to normal (mainly proto- and coproporphyrins found in the medium) while ethidium bromide-treated cells remain uroporphyric. The results suggest that impairment of mitochondrial functions in chicken by xenobiotics leads to uroporphyria development.

  19. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  20. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    Science.gov (United States)

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  1. Cholesterol Metabolism Is Altered in Rett Syndrome: A Study on Plasma and Primary Cultured Fibroblasts Derived from Patients

    Science.gov (United States)

    Segatto, Marco; Trapani, Laura; Di Tunno, Ilenia; Sticozzi, Claudia; Valacchi, Giuseppe; Hayek, Joussef; Pallottini, Valentina

    2014-01-01

    Rett (RTT) syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR), sterol regulatory element binding proteins (SREBPs), low density lipoprotein receptor (LDLr) and scavenger receptor B-1 (SRB-1) was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9) were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology. PMID:25118178

  2. Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome. Ultrastructural changes of mitochondria in cultured dermal fibroblasts of three patients.

    Science.gov (United States)

    Haust, M D; Dewar, R A; Gatfield, D P; Gordon, B A

    1996-03-01

    Mitochondria of fibroblasts cultured from the skin obtained at biopsy from three patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome, one of the autosomal recessive, heritable urea cycle disorders, were studied with appropriate controls ultrastructurally. The patients were two severely retarded 10- and 12-year-old boys, and a 22-year-old sister of the former whose mental status was at the low normal range; she never had motor impairments or seizures. The mitochondria, similar in all three patients, were increased in number, very long, branching and/or "looping," and tortuous. "Spurs" or "buddings" extended from their lateral surfaces and the terminal segments were often bulbous. Other unusual configurations were also present. In addition, giant forms with large diameter contained innumerable closely-packed and parallel cristae which traversed the entire width of these mitochondria; at times they assumed a "whirled" pattern. The mitochondrial matrix was usually of high electron density. These changes were not a feature of fibroblastic mitochondria of controls. Several changes resembled those of hepatic mitochondria in this disorder. All features are interpreted as an attempt at expanding the mitochondrial volume (via structural substratum) to compensate for the metabolic incompetence of these organelles (a block in transmembranous transfer of ornithine from hyaloplasm into mitochondria for conversion to citrulline).

  3. Cholesterol metabolism is altered in Rett syndrome: a study on plasma and primary cultured fibroblasts derived from patients.

    Directory of Open Access Journals (Sweden)

    Marco Segatto

    Full Text Available Rett (RTT syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2 are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR, sterol regulatory element binding proteins (SREBPs, low density lipoprotein receptor (LDLr and scavenger receptor B-1 (SRB-1 was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9 were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology.

  4. Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

    Science.gov (United States)

    Rufini, S; Cesaroni, M P; Balestro, N; Luly, P

    1996-12-01

    Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

  5. Shrink-induced biomimetic wrinkled substrates for functional cardiac cell alignment and culture.

    Science.gov (United States)

    Mendoza, Nicole; Tu, Roger; Chen, Aaron; Lee, Eugene; Khine, Michelle

    2014-01-01

    The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium, which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques, such as photolithography, generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition, such technologies are costly and require a clean room for fabrication. This chapter offers an easy, fast, robust, and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally, this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly, this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects.

  6. Compensatory function of bradykinin B1 receptor in the inhibitory effect of captopril on cardiomyocyte hypertrophy and cardiac fibroblast proliferation in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    ZOU Jun; REN Jiang-hua; FENG Dan; WANG Hong; XU Jiang

    2008-01-01

    Background Bradykinin(BK)acts mainly on two receptor subtypes:B1 and B2,and activation of B2 receptor mediates the most well-known cardioprotective effects of angiotensin converting enzyme inhibitors(ACEi),however,the role that B1 receptor plays in ACEi has not been fully defined.We examined the role of B1 receptor in the inhibitory effect of ACE inhibitor captopril on rat cardiomyocyte hypertrophy and cardiac fibroblast proliferation induced by angiotensin Ⅱ(Ang Ⅱ) and explored its possible mechanism.Methods Neonatal cardiomyocytes and cardiac fibroblasts(CFs)were randomly treated with Ang Ⅱ,captopril,B2 receptor antagonist(HOE-140)and B1 receptor antagonist(des-Arg10,Leu9-kallidin)alone or in combination.Flow cytometry was used to evaluate cell cycle,size and protein content.Nitric oxide(NO)and intracellular cyclic guanosine monophosphate(cGMP)level were measured by colorimetry and radioimmunoassay.Results After the CFs and cardiomyocytes were incubated with 0.1 μmol/L Ang Ⅱ for 48 hours.the percentage of CFs in the S stage,cardiomyocytes size and protein content significantly increased(both P<0.01 vs control),and these increases were inhibited by 10 μmol/L captopril.However,NO and cGMP levels were significantly higher than that with Ang Ⅱ alone(both P<0.01).1 μmol/L HOE-140 or 0.1 μmol/L des-Arg10,Leu9-kallidin attenuated the effects of captopril,which was blunted further by blockade of both B1 and B2 receptors.Conclusions Acting via B2 receptor,BK contributes to the antihypertrophic and antiproliferative effects of captopril on cardiomyocytes and CFs.In the absence of B2 receptor,B1 receptor may act a compensatory mechanism for the B2 receptor and contribute to the inhibition of cardiomyocyte hypertrophy and CFs proliferation by captopril.NO and cGMP play an important role in the effect of B1 receptor.

  7. Biological responses of three-dimensional cultured fibroblasts by sustained compressive loading include apoptosis and survival activity.

    Directory of Open Access Journals (Sweden)

    Toshiki Kanazawa

    Full Text Available Pressure ulcers are characterized by chronicity, which results in delayed wound healing due to pressure. Early intervention for preventing delayed healing due to pressure requires a prediction method. However, no study has reported the prediction of delayed healing due to pressure. Therefore, this study focused on biological response-based molecular markers for the establishment of an assessment technology to predict delayed healing due to pressure. We tested the hypothesis that sustained compressive loading applied to three dimensional cultured fibroblasts leads to upregulation of heat shock proteins (HSPs, CD44, hyaluronan synthase 2 (HAS2, and cyclooxygenase 2 (COX2 along with apoptosis via disruption of adhesion. First, sustained compressive loading was applied to fibroblast-seeded collagen sponges. Following this, collagen sponge samples and culture supernatants were collected for apoptosis and proliferation assays, gene expression analysis, immunocytochemistry, and quantification of secreted substances induced by upregulation of mRNA and protein level. Compared to the control, the compressed samples demonstrated that apoptosis was induced in a time- and load- dependent manner; vinculin and stress fiber were scarce; HSP90α, CD44, HAS2, and COX2 expression was upregulated; and the concentrations of HSP90α, hyaluronan (HA, and prostaglandin E2 (PGE2 were increased. In addition, the gene expression of antiapoptotic Bcl2 was significantly increased in the compressed samples compared to the control. These results suggest that compressive loading induces not only apoptosis but also survival activity. These observations support that HSP90α, HA, and, PGE2 could be potential molecular markers for prediction of delayed wound healing due to pressure.

  8. Transcriptional regulation of proteoglycans and glycosaminoglycan chain-synthesizing glycosyltransferases by UV irradiation in cultured human dermal fibroblasts.

    Science.gov (United States)

    Shin, Jeong-Eun; Oh, Jang-Hee; Kim, Yeon Kyung; Jung, Ji-Yong; Chung, Jin Ho

    2011-03-01

    Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm(2) of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, β1,3-glucuronyltransferase-1, β1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of β1,3-galactosyltransferase-6, β1,4-galactosyltransferase-3, -7, β-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

  9. Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures.

    Science.gov (United States)

    Masuki, Kouhei; Nomura, Yuji; Bhawal, Ujjal Kumar; Sawajiri, Masahiko; Hirata, Isao; Nahara, Yukinori; Okazaki, Masayuki

    2007-11-01

    The aim of this study was to examine the apoptotic and necrotic influence of four dental resin polymerization initiators--namely benzoyl peroxide (BPO), camphorquinone (CQ), dimethylaminoethyl methacrylate (DMAEMA), and dimethyl-para-toluidine (DMPT)--on human gingival fibroblast (HGF) cells. To this end, the growth inhibition of HGF cells with 1 mM BPO, CQ, and DMAEMA, and 500 microM DMPT was evaluated using Cell Counting Kit-8. Then, cell cycle analysis by flow cytometry was used to assess propidium iodide-stained cells (distribution of cells in G0/G1, S, G2/M phases). All four dental resin polymerization initiators induced G0/G1 cell cycle arrest. As for the patterns of cell death (necrosis and/or apoptosis), they were analyzed using Annexin V-FITC/PI staining with flow cytometry. All four dental resin polymerization initiators most likely induced necrosis.

  10. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    Science.gov (United States)

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  12. Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

    Science.gov (United States)

    Volpato, Luiz Evaristo Ricci; de Oliveira, Rodrigo Cardoso; Espinosa, Mariano Martinez; Bagnato, Vanderley Salvador; Machado, Maria A. A. M.

    2011-07-01

    Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm2), infrared laser (780 nm, 40 mW, 1 W/cm2), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm2). All applications were punctual and performed with a spot with 0.4 mm2 of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

  13. The role of basic fibroblast growth factor in glioblastoma multiforme and glioblastoma stem cells and in their in vitro culture.

    Science.gov (United States)

    Haley, Elizabeth M; Kim, Yonghyun

    2014-04-28

    Glioblastoma multiforme (GBM) is the most malignant form of central nervous system tumor, and current therapies are largely ineffective at treating the cancer. Developing a more complete understanding of the mechanisms controlling the tumor is important in order to explore new possible treatment options. It is speculated that the presence of glioblastoma stem or stem-like cells (GSCs), a rare type of pluripotent cancer cell that possesses the ability to self-renew and generate tumors, could be an important factor contributing to the resistance to treatment and deadliness of the cancer. A comprehensive knowledge of the mechanisms controlling the expression and properties of GSCs is currently lacking, and one promising area for further exploration is in the influence of basic fibroblast growth factor (FGF-2) on GSCs. Recent studies reveal that FGF-2 plays a significant part in regulating GBM, and the growth factor is commonly included as a supplement in media used to culture GSCs in vitro. However, the particular role that FGF-2 plays in GSCs has not been as extensively explored. Therefore, understanding how FGF-2 is involved in GSCs and in GBMs could be an important step towards a more complete comprehension of the managing the disease. In this review, we look at the structure, signaling pathways, and specific role of FGF-2 in GBM and GSCs. In addition, we explore the use of FGF-2 in cell culture and using its synthetic analogs as a potential alternative to the growth factor in culture medium.

  14. Effects of Basic Fibroblast Growth Factor and Insulin-like Growth Factor on Cultured Cartilage Cells from Skate Raja porasa

    Institute of Scientific and Technical Information of China (English)

    樊廷俊; 晋凌云; 汪小锋

    2003-01-01

    Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) on cartilage cells from proboscis of skate, Raja porasa Günther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24℃. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF-II at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-II was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF-II together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-II together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7.

  15. Fibroblast growth factor-1 and-2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures

    NARCIS (Netherlands)

    Yeoh, Joyce S. G.; van Os, Ronald; Weersing, Ellen; Ausema, Albertina; Dontje, Bert; Vellenga, Edo; de Haan, Gerald

    2006-01-01

    In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 + 2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assa

  16. Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts

    Directory of Open Access Journals (Sweden)

    Olsen Charlotta J

    2010-08-01

    Full Text Available Abstract Introduction Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. Methods In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s and cancer cells (MCF7S1 in three-dimensional (3D growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. Results In 3D co-culture both HMF3s and MCF7S1 cells demonstrated enhanced invasion into a Matrigel matrix. This was correlated with enhanced expression of the metastasis promoting S100A4 protein in fibroblasts, stimulation of the matrix metalloproteinase (MMP-2 activity, and enhanced secretion of a range of different cytokines. Orthotopic injection of oestrogen-dependent MCF7S1 cancer cells together with fibroblasts showed stimulation of tumour growth in mice without an external oestrogen supply. The resulting tumours were characterized by increased development of extracellular matrix, as well as an increase of murine S100A4 concentration and activity of MMP-2 in the tumour interstitial fluid. Conclusion Stimulation of the invasive phenotype of tumour cells in 3D co-cultures with fibroblasts could be correlated with increased production of S100A4 and MMP-2. We propose that enhanced development of mouse host-derived tumour stroma in a MCF7S1 co-injection xenograft model leads to oestrogen independency and is triggered by the

  17. Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M. van den; Heneweer, M.; Geest, M. de; Sanderson, T. [Inst. for Risk Assessment Sciences and Utrecht Univ. (Netherlands); Jong, P. de [St. Antonius Hospital, Nieuwegein (Netherlands); Bergman, A. [Stockholm Univ., Stockholm (Sweden)

    2004-09-15

    Methyl sulfonyl PCB metabolites (MeSO2-PCBs) are persistent contaminants and are ubiquitously present in humans and the environment. Lipophilicity of MeSO2- PCB metabolites is similar to the parent compounds and they have been detected in human milk, adipose, liver and lung tissue. 4- MeSO2-PCB-149 is the most abundant PCB metabolite in human adipose tissue and milk at a level of 1.5 ng/g lipids. Human blood concentration of 4-MeSO2-PCB-149 is approximately 0.03 nM. 3- MeSO2-PCB-101 is the predominant PCB metabolite in muscle and blubber in wildlife, such as otter, mink and grey seal. In the environment, they have been linked to chronic and reproductive toxicity in exposed mink. Additionaly, some MeSO{sub 2}-PCBs have been shown to be glucocorticoid receptor (GR) antagonists. Since approximately 60% of all breast tumors are estrogen responsive, exposure to compounds that are able to alter estrogen synthesis through interference with the aromatase enzyme, can lead to changes in estrogen levels and possibly to accelerated or inhibit breast tumor growth. Therefore, it is important to identify exogenous compounds that can alter aromatase activity in addition to those compounds which have direct interaction with the estrogen receptor (ER). Aromatase (CYP19) comprises the ubiquitous flavoprotein, NADPH-cytochrome P450 reductase, and a unique cytochrome P450 that is exclusively expressed in estrogen producing cells. Previous studies have revealed that expression of the aromatase gene is regulated in a species- and tissue specific manner. In healthy breast tissue, the predominantly active aromatase promoter region I.4 is regulated by glucocorticoids and class I cytokines. Therefore, it is important to investigate possible aromatase inhibiting properties of MeSO{sub 2}-PCBs (as anti glucocorticoids?) in relevant human tissues. We used primary human mammary fibroblasts because of their role in breast cancer development. We compared the results in primary fibroblasts with

  18. In vitro culture of ear fibroblast cells from Lu-chuan pig%陆川猪耳部成纤维细胞的体外培养

    Institute of Scientific and Technical Information of China (English)

    秦小娥; 胡林林; 卢晟盛; 卢克焕

    2011-01-01

    In order to establish an isolation method and culture system for Lu-chuan pig ear fibroblast cells,Lu-chuan pig ear fibroblast cell was isolated and cultured by tissue-piece methods.Using 0.25% trypsin + 0.02% EDTA as digested medium and DMEM containing 10% FBS as culture medium for subculture,it was concluded that culture system was good enough for lu-chuan pig ear fibroblast cells.The pured fibroblast cells could be obtained after passing 3 subcultures.The cultured cells were typical fibroblast morphology,which grooved as irregular triangle or shuttle,appearing as radioactive blaze and helix in shape.The cell type was identified by fluorescent immuneocytochemistry(ICC) and the result indicated that isolated cells from Lu-chuan ear tissues were Lu-chuan ear fibroblast cells.In conclusion,Lu-chuan pig ear fibroblast cell could be isolated and cultured by tissue-piece methods.%本研究建立陆川猪耳部成纤维细胞的体外培养体系,采用组织块培养法可以获得陆川猪耳部成纤维细胞。用0.25%胰蛋白酶+0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,能很好的支持陆川猪耳部成纤维细胞的生长。传3代后,观察到培养的细胞形态逐渐均一,为典型的成纤维细胞,绝大部分呈梭形或不规则三角形。细胞生长时呈放射状、火焰状或旋涡状走势。用间接免疫荧光法对细胞类型进行鉴定。抗波形蛋白免疫荧光染色显示为阳性,抗角形蛋白免疫荧光染色为阴性。结果,分离得到的细胞为成纤维细胞。结果表明,用组

  19. Trypanosoma acomys (Wenyon, 1909): continuous culturing with a mouse fibroblast cell-line (A9).

    Science.gov (United States)

    Abdallah, M A; Abdel-Hafez, S K; al-Yaman, F M

    1990-01-01

    The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.

  20. High content analysis of human fibroblast cell cultures after exposure to space radiation.

    Science.gov (United States)

    Dieriks, Birger; De Vos, Winnok; Meesen, Geert; Van Oostveldt, Kaat; De Meyer, Tim; Ghardi, Myriam; Baatout, Sarah; Van Oostveldt, Patrick

    2009-10-01

    Space travel imposes risks to human health, in large part by the increased radiation levels compared to those on Earth. To understand the effects of space radiation on humans, it is important to determine the underlying cellular mechanisms. While general dosimetry describes average radiation levels accurately, it says little about the actual physiological impact and does not provide biological information about individual cellular events. In addition, there is no information about the nature and magnitude of a systemic response through extra- and intercellular communication. To assess the stress response in human fibroblasts that were sent into space with the Foton-M3 mission, we have developed a pluralistic setup to measure DNA damage and inflammation response by combining global and local dosimetry, image cytometry and multiplex array technology, thereby maximizing the scientific output. We were able to demonstrate a significant increase in DNA double-strand breaks, determined by a twofold increase of the gamma-H2AX signal at the level of the single cell and a threefold up-regulation of the soluble signal proteins CCL5, IL-6, IL-8, beta-2 microglobulin and EN-RAGE, which are key players in the process of inflammation, in the growth medium.

  1. Ras Activated ERK and PI3K Pathways Differentially Affect Directional Movement of Cultured Fibroblasts

    Directory of Open Access Journals (Sweden)

    Leandra Sepe

    2013-01-01

    Full Text Available Background: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. Methods: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. Results: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. Conclusion: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement.

  2. Hormonal regulation of dipeptidyl-aminopeptidase I activity in cultured human fibroblasts.

    Science.gov (United States)

    Davis, M H

    1987-05-01

    Human male fibroblasts, cell line GM2987, were grown in 10% Nu-Serum or fetal bovine serum. Dipeptidyl-aminopeptidase I (DAP-I) activity was higher in cells grown with Nu-Serum and cells grown in 10% fetal bovine serum purchased from Grand Island Biological Company (GIBCO) and lower in cells grown in 10% fetal bovine serum obtained from Sterile Systems, Inc. (Hyclone). The addition of 0.3 microM cortisol to all three types of sera resulted in cells that had similar levels of DAP-I activity (maximum of 800-900 nmol of beta-naphthylamine released from glycyl-L-phenylanine-beta-naphthylamine per hour per milligram of cellular protein). The addition of cortisol to Hyclone fetal bovine serum increased the DAP-I levels by up to threefold with a half-maximal response occurring at 30 nM cortisol. Triiodothyronine also could increase DAP-I levels, but only between 1.5- and 2.0-fold. Testosterone propionate increased DAP-I levels by 1.4-fold. These changes in growth media and hormones had little effect on other lysosomal enzymes or the growth characteristics of the cells.

  3. Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

    Directory of Open Access Journals (Sweden)

    Hui-Xian Wang

    2017-07-01

    Full Text Available AIM: To explore the possibility of human umbilical cord mesenchymal stem cells (hUCMSCs, human umbilical vein endothelial cells (hUVECs, human dental pulp stem cells (hDPSCs and human periodontal ligament stem cells (hPDLSCs serving as feeder cells in co-culture systems for the cultivation of limbal stem cells. METHODS: Different feeder layers were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12 and were treated with mitomycin C. Rabbits limbal stem cells (LSCs were co-cultured on hUCMSCs, hUVECs, hDPSCs, hPDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency (CFE assay and immunofluorescence (IPO13,CK3/12. RESULTS: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But hUCMSCs, hDPSCs and hPDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to hUVECs and feeder-cell-free culture. CONCLUSION: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.

  4. Expression of MMP-2, MT1-MMP, and TIMP-2 by cultured rabbit corneal fibroblasts under mechanical stretch.

    Science.gov (United States)

    Liu, Chengxing; Feng, Pengfei; Li, Xiaona; Song, Jie; Chen, Weiyi

    2014-08-01

    Refractive surgery not only leads to tissue injury but also evokes mechanical stress increase of the cornea. How the mechanical stress affects the corneal matrix remodeling, specifically, matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases; TIMPs) is not well understood. In this study, cultured rabbit corneal fibroblasts in vitro were subjected to regimen of 5%, 10%, or 15% equibiaxial stretch at 0.1 Hz for 3 or 24 h. MMP-2 protein level was measured by gelatin zymography and Western blotting. MMP-2, membrane type 1 MMP (MT1-MMP), and TIMP-2 mRNA levels were quantified by real-time quantitative PCR. Extracellular regulated protein kinase (ERK) phosphorylation protein levels were assessed by Western blotting. Our results showed that a 15% stretch resulted in increases in MMP-2 protein, MMP-2 mRNA, and MT1-MMP mRNA levels, but a decrease in TIMP-2 mRNA level. However, a 5% stretch caused decreases in MMP-2 protein and mRNA level, but an increase in TIMP-2 mRNA level, and no change in MT1-MMP mRNA level. A 15% stretch also caused a significant increase in ERK1/2 phosphorylation. Inhibition of the mitogenactivated protein kinase (MEK) pathway with PD98059 attenuated stretch-induced increase in MMP-2 production and ERK activity. These results suggest that small-magnitude stretching may promote corneal matrix synthetic events, whereas large-magnitude stretching promotes corneal matrix degradation by changing the balance between MMPs and TIMPs in corneal fibroblasts. Large-magnitude stretch-induced increase in pro-MMP-2 production was in an ERK-dependent manner. © 2014 by the Society for Experimental Biology and Medicine.

  5. Effects of serum from patients with atrial fibrillation on chemotaxis of cardiac fibroblasts%房颤患者血清对心肌成纤维细胞趋化运动的影响

    Institute of Scientific and Technical Information of China (English)

    段卡丹; 李裕舒; 史晓婧

    2011-01-01

    目的:研究心肌成纤维细胞的运动特性,以及房颤与非房颤患者血清对成纤维细胞趋化运动诱导能力的差异.方法:用胰酶和胶原酶消化、差速贴壁法培养新生SD大鼠的心肌成纤维细胞,取3-4代采用Transwell装置检测房颤组血清与非房颤组血清诱导成纤维细胞运动的能力差异.结果:在各组血清的趋化诱导作用下,与对照组相比,迁移至下室的细胞数明显增多.其中持续性房颤组最多,非房颤房性心律失常组明显高于阵发性房颤组,对照组细胞数最少,各组比较均具有显著差异.Logistic回归分析表明,迁移至滤膜下的细胞数与左房直径以及房颤有相关性.结论:(1)各组血清的趋化诱导能力高于对照组,不同组间的差异说明心房纤维化是一个慢性、隐匿、迁延的过程.以修复为目的的成纤维细胞迁移浸润数量间接反映了心肌损伤的存在、范围和程度.(2)迁移浸润的细胞数变化先于左房直径变化,提示房颤和左房直径增大的正相关关系可能并非直接的因果关系,心房肌损伤后的纤维化可能参与其中.%AIM: To investigate the effects of the serum from the patients with atrial fibrillation ( AF) on the chemotaxis of rat cardiac fihroblasts.METHODS : Cardiac fibroblasts were isolated from the ventricles of neonatal Sprague - Dawley rats and primarily cultured with digestion and differential adhesion.The cells in 3 to 4 passages were used for Transwell chamber assay to determine the chemotatic effects of the sera.RESULTS: Compared with control group, the cells that migrated through the polycarbonate membrane were obviously increased in AF group.The strongest chemotaxis was induced by the serum from the patients with persistent atrial fibrillation( pers - AF) .The number of migrated cells in non - AF atrial arrhythmia( AA) group was higher than that in paroxysmal atrial fihrillation( paro - AF) group, and that in control group was the

  6. Multiple Directional Differentiation Difference of Neonatal Rat Fibroblasts from Six Organs

    Directory of Open Access Journals (Sweden)

    Yuqiao Chang

    2016-06-01

    Full Text Available Background/Aims: Fibroblasts are abundantly distributed throughout connective tissues in the body and are very important in maintaining the structural and functional integrity. Recent reports have proved that fibroblasts and mesenchymal stem cells share much more in common than previously recognized. The aim of this study was to investigate comparative studies in fibroblasts on the differences in the expression of molecular markers and differentiation capacity from different organs. Methods: Combined trypsin/collagenase enzymes digestion method was used to isolate and culture the fibroblasts derived from heart, liver, spleen, lung, kidney and skin. Cell activity was determined by methyl thiazolyl tetrazolium (MTT assay. Common molecular markers for fibroblasts such as vimentin, DDR2 and FSP1, stem cell markers nanog, c-kit and sca-1 were detected by RT-PCR, immunofluorescence and western blotting. The osteogenic, adipogenic and cardiogenic differentiations of fibroblasts were performed by inductive culture in special mediums, and analyzed by Alizarin red, Oil red O and immunofluorescence staining of cTnT respectively. Results: The proliferation rate of fibroblasts in lung was faster than in other five organs. Common molecular markers for fibroblasts were expressed differently in different organs. DDR2 was strongly expressed in fibroblasts in the heart, partly expressed in the heart, skin, liver and spleen. Interestingly, no expression of DDR2 was detected in liver and kidney. However, vimentin and FSP1 were consistently expressed in fibroblasts from skin, liver, kidney, spleen and lung. nanog expression in fibroblasts from lung was less than that from heart, skin, liver and spleen (P . c-kit expression in fibroblasts from heart, skin and kidney was higher than that from spleen (P , while the c-kit positive fibroblasts from liver was obviously higher than that from spleen (P . But sca-1 expression in fibroblasts from lung was the lowest among six

  7. The differential proliferative response of fetal and adult human skin fibroblasts to TGF-β is retained when cultured in the presence of fibronectin or collagen.

    Science.gov (United States)

    Armatas, Andreas A; Pratsinis, Harris; Mavrogonatou, Eleni; Angelopoulou, Maria T; Kouroumalis, Anastasios; Karamanos, Nikos K; Kletsas, Dimitris

    2014-08-01

    Transforming growth factor-β is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-β inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-β on fetal and adult cells. Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-β was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. Fetal skin fibroblast-proliferation was inhibited by TGF-β, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-β on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-β when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. The interplay between TGF-β and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Poly(l-lactic acid) and polyurethane nanofibers fabricated by solution blow spinning as potential substrates for cardiac cell culture.

    Science.gov (United States)

    Tomecka, Ewelina; Wojasinski, Michal; Jastrzebska, Elzbieta; Chudy, Michal; Ciach, Tomasz; Brzozka, Zbigniew

    2017-06-01

    This paper presents a comparison and evaluation of cardiac cell proliferation on poly(l-lactic acid) (PLLA) and polyurethane (PU) nanofibrous mats fabricated by solution blow spinning (SBS). Three different cardiac cell lines: rat cardiomyoblasts (H9C2 line), human (HCM) and rat cardiomyocytes (RCM) were used for experiments. Cell morphology, orientation and proliferation were investigated on non-modified and protein-modified (fibronectin, collagen, gelatin, laminin, poly-l-lysine) surfaces of both types of nanofibers. Obtained results of cell culture on nanofibers surfaces were compared to the results of cell culture on polystyrene (PS) surfaces modified in the same way. The results indicated that in most cases polymeric nanofibers (PLLA and PU) are better substrates for cardiac cell culture than PS surfaces. All types of investigated cells, cultured on nanofibers (PLLA and PU), had more elongated shape than cells cultured on PS surfaces. Moreover, cells were arranged in parallel to each other, according to fibers orientation. Additionally, it was shown that the protein modifications of investigated surfaces influenced on cell proliferation. Therefore, we suggest that the cardiac cell culture on nanofibrous mats fabricated by SBS could be more advanced experimental in vitro model for studies on the effect of various cardiac drugs than traditional culture on PS surface. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

    Science.gov (United States)

    Boemi, L; Allison, G M; Graham, W P; Krummel, T M; Ehrlich, H P

    1999-10-01

    Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.

  10. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture.

    Science.gov (United States)

    Gilhare, Varsha Rani; Hirpurkar, S D; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

    2015-03-01

    The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-15

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

  12. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    Science.gov (United States)

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  13. [Effects of Bushen Shuji Granule on IL-6 in the culture fluid sample of fibroblast cells from the synovial liquid of the ankylosing spondylitis patients].

    Science.gov (United States)

    Zhu, Jun-Ling; Yan, Xiao-Ping

    2012-02-01

    To explore the effects of Bushen Shuji Granule (BSG) on inhibiting the interleukin 6 (IL-6) level in the synovial fluid sample of fibroblast cells from the ankylosing spondylitis (AS) patients. Using serum pharmacologic method, the IL-6 level in the culture fluid sample of fibroblast cells was observed by ELISA method with different concentrations of medicated serum containing BSG. The IL-6 level at the mRNA level was detected using reverse transcriptase-polymerase chain reaction (RT-PCR). The vehicle serum and sulfasalazine (SSZ) serum were taken as controls. Results of ELISA showed the IL-6 level in the AS group was more than that in the vehicle serum group with obvious statistical difference. BSG could obviously inhibited the IL-6 level, showing statistical difference when compared with the vehicle serum group. Besides, obvious dose-dependent correlation existed between BSG and its inhibition on fibroblast proliferation. And the IL-6 level at the mRNA level in the AS group was higher than that in the vehicle serum group, showing statistical difference by semi-quantitative analysis. BSG could play its clinical role of anti-inflammation and anti-fibrosis through inhibiting the IL-6 level in the culture fluid sample of fibroblast cells.

  14. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  15. The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs as a co-culture in vitro

    Directory of Open Access Journals (Sweden)

    Król Magdalena

    2012-03-01

    Full Text Available Abstract Background It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs in vitro. Results A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p Conclusion The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

  16. Human ovarian fibroblasts culture%人正常卵巢组织中成纤维细胞的培养

    Institute of Scientific and Technical Information of China (English)

    董丽华; 孙玮; 傅士龙; 韩素萍

    2011-01-01

    Objective; To obtain the purified human ovarian normal fibroblasls (NFs) preliminarily. Methods; The ovarian tissues were dissociated by 0.25% trypsin, after overgrowing on the bottom of the culture dish,the cells were adhered to the bottom of the dish repeatedly to depurate them. Then the cells were verified according to its morphology under the inverted-microscope and were further confined by a series of biological markers with immunocytochemistry. And its. Growth cure was observed. Results; The typical shape of ovarian NFs was fusiform, cords like,triangular,polyangular and irregular. Most of them contain Pseudopods-like structure. The NFs showed positive staining for vimentin.fibroblast specific protein-1, desmin and a-smooth muscle actin,negative staining for cytokeratinpan,cytokeratin7, cytokeratin 20, CD31, CD90, MMP-2, MMP-3, MMP-9, CA125, CA19-9, CEA, fibroblast activation proteina (FAP-a) and platelet-derived growth factor receptora (PDGFR-a). Conclusion; Human ovarian NFs were cultured successfully, which provides a tool for further studies on ovarian cancer and carcinoma associated fibroblasts.%目的:通过分离、纯化、培养及鉴定,获得典型人卵巢成纤维细胞.方法:采用酶消化+反复贴壁法,获得纯化成纤维细胞,通过形态学观察和免疫细胞化学染色,对所得细胞进行特征性标志及生物学特性的检测,进一步阐述细胞特征,并绘制细胞生长曲线.结果:形态学上,正常卵巢成纤维细胞形态多样,大部分为梭形、条索形,还有三角形、多角形和不规则形等,并具有伪足样外形;其生长潜伏期较长,12~14 d可传代;免疫细胞化学上,波形蛋白(vimentin)表达强阳性,结蛋白(desmin)、成纤维细胞特异性蛋白1(FSP-1)表达弱阳性;在较早期传代的细胞中,CK-7及CK-P呈少量阳性,随着细胞的纯化,其阳性表达渐行减少直至完全消失;肌动蛋白α(α-SMA)部分细胞表达阳性.而卵巢黏液性细胞标志CK-20

  17. Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

    Science.gov (United States)

    Mytych, Jennifer; Wnuk, Maciej; Rattan, Suresh I S

    2016-04-01

    Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells.

  18. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Yoshino A

    2016-08-01

    Full Text Available Atsushi Yoshino,1 Natalia Polouliakh,1–3 Akira Meguro,1 Masaki Takeuchi,1,4 Tatsukata Kawagoe,1 Nobuhisa Mizuki1 1Department of Ophthalmology and Visual Science, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 2Sony Computer Science Laboratories Inc., Fundamental Research Laboratories, 3Systems Biology Institute, Tokyo, Japan; 4Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA Abstract: Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. Keywords: fish egg, antiaging, gene expression analysis, antioxidative gene, phylogenetic footprinting analysis

  19. Notch Signaling Mediates TNF-α-Induced IL-6 Production in Cultured Fibroblast-Like Synoviocytes from Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Zhijun Jiao

    2012-01-01

    Full Text Available It has been reported that Notch family proteins are expressed in synovium tissue and involved in the proliferation of synoviocyte from rheumatoid arthritis (RA. The aim of this paper was to investigate whether Notch signaling mediated TNF-α-induced cytokine production of cultured fibroblast-like synoviocytes (FLSs from RA. Exposure of RA FLSs to TNF-α (10 ng/ml led to increase of Hes-1, a target gene of Notch signaling, and a marked upregulation of Notch 2, Delta-like 1, and Delta-like 3 mRNA levels. Blockage of Notch signaling by a γ-secretase inhibitor (DAPT inhibited IL-6 secretion of RA FLSs in response to TNF-α while treatment with recombinant fusion protein of Notch ligand Delta-like 1 promoted such response. TNF-α stimulation also induced IL-6 secretion in OA FLSs; however, the Hes-1 level remained unaffected. Our data confirm the functional involvement of Notch pathway in the pathophysiology of RA FLSs which may provide a new target for RA therapy.

  20. Diminished internalization and action of 1,25-dihydroxyvitamin D3 in dermal fibroblasts cultured from New World primates

    Energy Technology Data Exchange (ETDEWEB)

    Adams, J.S.; Gacad, M.A.; Baker, A.J.; Kheun, G.; Rude, R.K.

    1985-06-01

    We investigated the occurrence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-resistant osteomalacia in the New World primate colony of Saguinus imperator at the Los Angeles Zoo. The mean serum concentration of 1,25-(OH)2D3 was elevated 5-fold in the New World primates compared to that in their Old World counterparts. The specific internalization of 0.6 nM (/sup 3/H)1,25-(OH)2D3 by cultured dermal fibroblasts from New World primates was reduced 75% compared to that by cells from Old World primates or man. The decrease in hormone uptake resulted from a decrease in the number of high affinity intracellular binding sites for 1,25-(OH)2D3 and apparently caused a 90-95% reduction in 1,25-(OH)2D3-induced 25-hydroxyvitamin-D3-24-hydroxylase activity. There was no alteration in the capacity or avidity of New World primate serum for 1,25-(OH)2D3 compared to that of serum from Old World primates. These data suggest that the occurrence of vitamin D-resistant osteomalacia in New World primates is the result of decreased high affinity, receptor-mediated uptake of 1,25-(OH)2D3 by the target cell.

  1. Investigation of Adaptive Responses in Bystander Cells in 3D Cultures Containing Tritium-Labeled and Unlabeled Normal Human Fibroblasts

    Science.gov (United States)

    Pinto, Massimo; Azzam, Edouard I.; Howell, Roger W.

    2010-01-01

    The study of radiation-induced bystander effects in normal human cells maintained in three-dimensional (3D) architecture provides more in vivo-like conditions and is relevant to human risk assessment. Linear energy transfer, dose and dose rate have been considered as critical factors in propagating radiation-induced effects. This investigation uses an in vitro 3D tissue culture model in which normal AG1522 human fibroblasts are grown in a carbon scaffold to investigate induction of a G1 arrest in bystander cells that neighbor radiolabeled cells. Cell cultures were co-pulse-labeled with [3H]deoxycytidine (3HdC) to selectively irradiate a minor fraction of cells with 1–5 keV/μm β particles and bromodeoxyuridine (BrdU) to identify the radiolabeled cells using immunofluorescence. The induction of a G1 arrest was measured specifically in unlabeled cells (i.e. bystander cells) using a flow cytometry-based version of the cumulative labeling index assay. To investigate the relationship between bystander effects and adaptive responses, cells were challenged with an acute 4 Gy γ-radiation dose after they had been kept under the bystander conditions described above for several hours, and the regulation of the radiation-induced G1 arrest was measured selectively in bystander cells. When the average dose rate in 3HdC-labeled cells (bystander effects or adaptive bystander effects were observed as measured by magnitude of the G1 arrest, micronucleus formation, or changes in mitochondrial membrane potential. Higher dose rates and/or higher LET may be required to observe stressful bystander effects in this experimental system, whereas lower dose rates and challenge doses may be required to detect adaptive bystander responses. PMID:20681788

  2. Biocompatibility of potential wound management products: fungal mycelia as a source of chitin/chitosan and their effect on the proliferation of human F1000 fibroblasts in culture.

    Science.gov (United States)

    Chung, L Y; Schmidt, R J; Hamlyn, P F; Sagar, B F; Andrews, A M; Turner, T D

    1994-04-01

    Aspergillus oryzae, Mucor mucedo, and Phycomyces blakesleeanus cultures were examined as sources of chitin/chitosan. The nitrogen content of the alkali-treated mycelia/sporangiophores of A. oryzae, M. mucedo, and P. blakesleeanus was 2.52, 3.61, and 6.27% w/w, which relates to an estimated chitin content of 37, 52, and 91%, respectively. The effect of these fungal materials on the rate of proliferation of human F1000 fibroblasts in culture was examined. At 0.01% w/v, all three materials exhibited significant (P mucedo produced a significant (P mucedo, possessed cell attractant properties, again correlating with chitin content. If developed for use as wound management materials, the sporangiophores of P. blakesleeanus and the mycelium of M. mucedo could possibly promote the growth of fibroblasts and provide a matrix for their anchorage, thus contributing to the granulation phase of the healing cascade.

  3. Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co-culture models of cells.

    Science.gov (United States)

    Jarkovska, Karla; Dvorankova, Barbora; Halada, Petr; Kodet, Ondrej; Szabo, Pavol; Gadher, Suresh Jivan; Motlik, Jan; Kovarova, Hana; Smetana, Karel

    2014-07-01

    The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns. We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation. The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  4. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  5. Action of SNAIL1 in Cardiac Myofibroblasts Is Important for Cardiac Fibrosis following Hypoxic Injury

    Science.gov (United States)

    Biswas, Hirak; Longmore, Gregory D.

    2016-01-01

    Hypoxic injury to the heart results in cardiac fibrosis that leads to cardiac dysfunction and heart failure. SNAIL1 is a zinc finger transcription factor implicated in fibrosis following organ injury and cancer. To determine if the action of SNAIL1 contributed to cardiac fibrosis following hypoxic injury, we used an endogenous SNAIL1 bioluminescence reporter mice, and SNAIL1 knockout mouse models. Here we report that SNAIL1 expression is upregulated in the infarcted heart, especially in the myofibroblasts. Utilizing primary cardiac fibroblasts in ex vivo cultures we find that pro-fibrotic factors and collagen I increase SNAIL1 protein level. SNAIL1 is required in cardiac fibroblasts for the adoption of myofibroblast fate, collagen I expression and expression of fibrosis-related genes. Taken together this data suggests that SNAIL1 expression is induced in the cardiac fibroblasts after hypoxic injury and contributes to myofibroblast phenotype and a fibrotic scar formation. Resultant collagen deposition in the scar can maintain elevated SNAIL1 expression in the myofibroblasts and help propagate fibrosis. PMID:27706205

  6. In vitro effects of Choukroun's PRF (platelet-rich fibrin) on human gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts in primary cultures.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Diss, Antoine; Odin, Guillaume; Doglioli, Pierre; Hippolyte, Marie-Pascale; Charrier, Jean-Baptiste

    2009-09-01

    The objective of this study was to analyze the effects of Choukroun's PRF (platelet-rich fibrin), a leucocyte and platelet concentrate clinically usable as fibrin membrane or clot, on human primary cultures of gingival fibroblasts, dermal prekeratinocytes, preadipocytes, and maxillofacial osteoblasts. For the proliferation study, these cells were cultivated with or without a PRF membrane originating from the same donor as for the cells. For osteoblasts and fibroblasts, dose-dependent effect was assessed (using 2 membranes). Cell counts and cytotoxicity tests were performed at 3, 7, 14, and 21 days, and even 28 days for osteoblasts. More osteoblast cultures were prepared in differentiation conditions according to 3 modalities (without PRF, with PRF, with PRF the first day and differentiation medium applied only after the first week of culture). Osteoblast differentiation was analyzed using Von Kossa staining and alkaline phosphatase, DNA and total cell proteins dosage. PRF induced a significant and continuous stimulation of proliferation in all cell types. It was dose dependent during all the experiment with osteoblasts, but only on day 14 with fibroblasts. Moreover, PRF induced a strong differentiation in the osteoblasts, whatever the culture conditions. The analysis of osteoblast cultures in differentiation conditions with PRF, using light and scanning electron microscopy, revealed a starting mineralization process in the PRF membrane itself after 14 days. Moreover, PRF leucocytes seemed to proliferate and interact with osteoblasts. Cultures with PRF are always cocultures with leucocytes. These "chaperone leucocytes" could be the source of differential geographic regulation within the culture and explain the double contradictory effect proliferation/differentiation observed on osteoblasts.

  7. Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

    Science.gov (United States)

    Nakanishi, Masanori; Sato, Tadashi; Nelson, Amy J.; Farid, Maha; Michalski, Joel; Kanaji, Nobuhiro; Wang, Xingqi; Basma, Hesham; Patil, Amol; Goraya, Jadvinder; Liu, Xiangde; Togo, Shinsaku; L. Toews, Myron; Holz, Olaf; Muller, Kai-Christian; Magnussen, Helgo

    2012-01-01

    Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E2 (PGE2) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE2 of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbecco's modified Eagle's medium, with or without PGE2, and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE2 and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE2 was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase–A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE2. The increased release of VEGF induced by PGE2 was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE2 can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall. PMID:22298530

  8. Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury

    OpenAIRE

    Jimenez, Ana Gabriela; Harper, James M.; Simon A. Queenborough; Williams, Joseph B.

    2013-01-01

    A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cel...

  9. Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture.

    Science.gov (United States)

    Kondo, Yukiko; Fukuda, Ken; Adachi, Tadafumi; Nishida, Teruo

    2008-11-01

    Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-kappaB and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-kappaB activation is mediated by phosphorylation of the inhibitor IkappaB by IkappaB kinase (IKK)-2 and consequent IkappaB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IkappaBalpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. IL-1beta-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration- and time-dependent manner. TPCA-1 inhibited the IL-1beta-induced expression of MMP-1, -3, and -9 in these cells at both the mRNA and protein levels and the IL-1beta-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IkappaBalpha and the nuclear translocation of NF-kappaB induced by IL-1beta. An IKK-2 inhibitor blocked IL-1beta-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-kappaB signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.

  10. 西红花酸对心肌成纤维细胞增殖和胶原合成的影响%Effects of crocetin on the proliferation and collagen synthesis of cardiac fibroblasts

    Institute of Scientific and Technical Information of China (English)

    胡慧; 钱之玉; 成文媛; 杨丽娜; 朱艳虹; 陶赟

    2012-01-01

    AIM: To investigate the effects of crocetin on proliferation and collagen synthesis in cultured neonatal rat cardiac fibroblasts (CFB) stimulated by angiotensin II (Ang II )? and to explore the action mechanism of crocetin. METHODS: We used cultured cardiac fibroblasts from neonatal 1-3 days rat and treating with Ang II to induce fibrosis model. The effects of crocetin on proliferation of CFB were observed by MTT assay, synthesis of collagen was observed by the hydroxyproline concentration measured. Cell cyclin distribution was determined with flow cytometre. Collagen I , collagen El , matrix metalloproteinase (MMP-2), the tissue inhibitor metalloproteinase (TIMP-1) and TGF-β, mRNA expression were examined by Q-PCR. The expression of P27 was tested by Western blotting. RESULTS; Within a concentration coverage, crocetin inhibited CFB proliferation and collagen synthesis induced by Ang II in a dose-dependent manner. The percentage of cells in G0 /G1 phase in crocetin groups increased with concentration, the percentage of cells in S and G2/M phases and the proliferation index in crocetin groups decreased with concentration, which compared with Ang I] group difference was statistically significant (P<0.05, P<0. 01, respectively). Crocetin decreased the levels of Collagen I mRNA, Collagen III mRNA, TIMP1 mRNA and TGF-β1 mRNA expression, but increased MMP-2 mRNA expression. ④ Crocetin decreased the protein expression of P27 stimulated by Ang II . CONCLUSION: Crocetin inhibited CFB proliferation and collagen synthesis stimulated by Ang TJ , which related to the cytokines excreting and the regulation of extracellular matrix.%目的:探讨西红花酸(crocetin)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌成纤维细胞(cardiac fibroblasts,CFB)增殖和胶原合成的影响及其作用机制.方法:体外培养新生SD大鼠CFB,建立AngⅡ诱导新生大鼠CFB纤维化模型;MTT法检测CFB的增殖;羟脯氨酸测定检测CFB胶原含量;流

  11. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  12. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  13. Complex-periodic spiral waves in confluent cardiac cell cultures induced by localized inhomogeneities

    Science.gov (United States)

    Hwang, Seong-min; Kim, Tae Yun; Lee, Kyoung J.

    2005-07-01

    Spatiotemporal wave activities in excitable heart tissues have long been the subject of numerous studies because they underlie different forms of cardiac arrhythmias. In particular, understanding the dynamics and the instabilities of spiral waves have become very important because they can cause reentrant tachycardia and their subsequent transitions to fibrillation. Although many aspects of cardiac spiral waves have been investigated through experiments and model simulations, their complex properties are far from well understood. Here, we show that intriguing complex-periodic (such as period-2, period-3, period-4, or aperiodic) spiral wave states can arise in monolayer tissues of cardiac cell culture in vitro, and demonstrate that these different dynamic states can coexist with abrupt and spontaneous transitions among them without any change in system parameters; in other words, the medium supports multistability. Based on extensive image data analysis, we have confirmed that these spiral waves are driven by their tips tracing complex orbits whose unusual, meandering shapes are formed by delicate interplay between localized conduction blocks and nonlinear properties of the culture medium. Author contributions: S.-m.H. and K.J.L. designed research; S.-m.H. and T.Y.K. performed research; S.-m.H. contributed new reagents/analytic tools; S.-m.H., T.Y.K., and K.J.L. analyzed data; and S.-m.H. and K.J.L. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Abbreviations: IBI, interbeat interval; P-n, period-n.

  14. Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment.

    Science.gov (United States)

    Kim, Yong Sook; Jeong, Hye-Yun; Kim, Ah Ra; Kim, Woong-Hee; Cho, Haaglim; Um, JungIn; Seo, Youngha; Kang, Wan Seok; Jin, Suk-Won; Kim, Min Chul; Kim, Yong-Chul; Jung, Da-Woon; Williams, Darren R; Ahn, Youngkeun

    2016-08-11

    The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2'Z,3'E)-6-Bromoindirubin-3'-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.

  15. Surface modified superparamagnetic nanoparticles: Interaction with fibroblasts in primary cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Chapa Gonzalez, Christian; Roacho Pérez, Jorge A.; Martínez Pérez, Carlos A.; Olivas Armendáriz, Imelda [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Jimenez Vega, Florinda [Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente del PRONAF y Estocolmo, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Castrejon Parga, Karen Y. [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Garcia Casillas, Perla E., E-mail: pegarcia@uacj.mx [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico)

    2014-12-05

    Highlights: • An inorganic layer before an organic material shell onto MNPs improves cell viability. • The coating type and the concentration of nanoparticles directly affect cell viability. • Modified magnetite nanoparticles with organic and inorganic materials was developed. - Abstract: The development of a variety of medical applications such as drug delivery, cell labeling, and medical imaging have been possible owing to the unique features exhibited by magnetic nanoparticles. Nanoparticle–cell interaction is related to the surface aspects of nanoparticle, which may be described based on their chemistry or inorganic/organic characteristics. The coating on particle surface reduces the inter-particle interactions and provides properties such as biocompatibility. Among the coating materials used for nanoparticles employed in biomedical applications, oleic acid is one of the most utilized due to its biocompatibility. However, a major drawback with this naturally occurring fatty acid is that it is easily oxidized by cells and this reduces their performance in biomedical applications. In order to avoid the direct contact of the cell with the magnetite particle, coating with an inorganic material prior to the oleic acid shell would be effective. This would retard the magnetite dissociation thereby improve the cell viability. Here we report our investigation on the effect of surface modified magnetite nanoparticles (MNPs) on the cell viability using primary cultures incubated with those particles. We prepared magnetite nanoparticles by chemical co-precipitation method; nanoparticle surface was first modified by silanol condensation followed by chemisorption of oleic acid. All nanostructures have a particle size less than 100 nm, depending on the material coating and superparamagnetic behavior. The saturated magnetizations (M{sub s}) of the magnetite samples coated with oleic acid (MAO; 49.15 emu/g) and double shell silica-oleic acid (MSAO; 46.16 emu/g) are

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

    DEFF Research Database (Denmark)

    Almqvist, Sofia; Werthén, Maria; Johansson, Anna

    2010-01-01

    or down-regulation of genes, of which most are involved in cellular growth, migration and differentiation. The effect of amelogenin was exemplified by increased proliferation over 7 days. In conclusion, the beneficial effects of amelogenin on wound healing are possibly conducted by stimulating fibroblast......Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100...... signalling, proliferation and migration via integrin interactions. It is hypothesized that amelogenin stimulates wound healing by providing connective tissue cells with a temporary extracellular matrix....

  17. Feasibility analysis of using MPC30-DEA70 to transfect microRNA-21 AS-ODN into cardiac fibroblasts%MPC30-DEA70载 microRNA-21AS-ODN转染心肌成纤维细胞的可行性分析

    Institute of Scientific and Technical Information of China (English)

    瞿晓雅; 李艳茹; 张琼; 张卓琦

    2015-01-01

    Objective To investigate whether a cationic phosphorylcholine polymer MPC 30-DEA70 ( PC polymer ) can transfect the microRNA-21 antisense oligonucleotide ( AS-ODN) into the rat cardiac fibroblasts in vitro successfully , and then to observe the effects of MPC30-DEA70/microRNA-21ODN complexes on the expression of pre-microRNA-21 and the inhibitory effects on downstream products in rat cardiac fibroblasts after transfection .Methods MPC30-DEA70 was com-plexed with microRNA-21ODN at different N∶P ratios.Cardiac fibroblasts from neonatal Sprague Dawley (SD) rats were i-solated by trypsin digestion and cultured by differential attachment methods .The distribution of MPC 30-DEA70/microRNA-21ODN complexes in the cells was observed by fluorescence microscopy ( FM) and confocal laser scanning microscopy (CLSM).The expression of ERK-MAP kinase and collagenⅠproteins and pre-microRNA-21 was detected by Western blot-ting and RT-PCR.Results Under the inverted microscope , the primary cultured cardiac fibroblasts appeared irregular tri-angle and polygon, and oval nuclei in the middle.The transfection efficiency of MPC30-DEA70/microRNA-21ODN comple-xes was high in the cardiac fibroblasts, and was in a dosage-dependent manner (all P<0.05).From the CLSM images, most of the complexes located around the cell nucleus and a few were in inside of the nucleus .When MPC30-DEA70 trans-fected the microRNA-21ODN into the cardiac fibroblasts , the expression of pre-microRNA-21, ERK-MAP kinase and colla-genⅠproteins was significantly down-regulated (all P<0.05).Conclusions MPC30-DEA70 may transfect the microRNA-21ODN into the cardiac fibroblasts in vitro successfully , and the expression of microRNA-21, ERK-MAP kinase and colla-genⅠproteins is significantly down-regulated.Thus, MPC30-DEA70 can work as an effective transgenic vector .%目的:探讨阳离子磷酸胆碱聚合物MPC30-DEA70能否成功载microRNA-21反义寡核苷酸(AS-ODN)转染大鼠心肌成纤

  18. 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.

  19. Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts

    Science.gov (United States)

    Gao, Song; Chen, Keyang; Zhao, Yinzhi; Rich, Celeste B.; Chen, Lijun; Li, Sandy J.; Toselli, Paul; Stone, Phillip; Li, Wande

    2005-01-01

    Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0–120 μg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80–120 μg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 μg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 μg CSC/ml. Moreover, 80–120 μg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema. PMID:15933228

  20. Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Mami; Inoue, Takahiro; Shirai, Takuma; Takamatsu, Kazuhiko; Kunihiro, Shiori; Ishii, Hirokazu [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Nishikata, Takahito, E-mail: nisikata@konan-u.ac.jp [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe 650-0047 (Japan)

    2014-07-31

    The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

  1. Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Rachel C Williams

    Full Text Available Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival

  2. Highly cytocompatible and flexible three-dimensional graphene/polydimethylsiloxane composite for culture and electrochemical detection of L929 fibroblast cells.

    Science.gov (United States)

    Waiwijit, Uraiwan; Maturos, Thitima; Pakapongpan, Saithip; Phokharatkul, Ditsayut; Wisitsoraat, Anurat; Tuantranont, Adisorn

    2016-08-01

    Recently, three-dimensional graphene interconnected network has attracted great interest as a scaffold structure for tissue engineering due to its high biocompatibility, high electrical conductivity, high specific surface area and high porosity. However, free-standing three-dimensional graphene exhibits poor flexibility and stability due to ease of disintegration during processing. In this work, three-dimensional graphene is composited with polydimethylsiloxane to improve the structural flexibility and stability by a new simple two-step process comprising dip coating of polydimethylsiloxane on chemical vapor deposited graphene/Ni foam and wet etching of nickel foam. Structural characterizations confirmed an interconnected three-dimensional multi-layer graphene structure with thin polydimethylsiloxane scaffold. The composite was employed as a substrate for culture of L929 fibroblast cells and its cytocompatibility was evaluated by cell viability (Alamar blue assay), reactive oxygen species production and vinculin immunofluorescence imaging. The result revealed that cell viability on three-dimensional graphene/polydimethylsiloxane composite increased with increasing culture time and was slightly different from a polystyrene substrate (control). Moreover, cells cultured on three-dimensional graphene/polydimethylsiloxane composite generated less ROS than the control at culture times of 3-6 h. The results of immunofluorescence staining demonstrated that fibroblast cells expressed adhesion protein (vinculin) and adhered well on three-dimensional graphene/polydimethylsiloxane surface. Good cell adhesion could be attributed to suitable surface properties of three-dimensional graphene/polydimethylsiloxane with moderate contact angle and small negative zeta potential in culture solution. The results of electrochemical study by cyclic voltammetry showed that an oxidation current signal with no apparent peak was induced by fibroblast cells and the oxidation current at an

  3. 人成纤维细胞的原代培养及鉴定%Primary culture and identification of human fibroblasts

    Institute of Scientific and Technical Information of China (English)

    鲍荣辉; 刘先哲; 周军

    2011-01-01

    目的 建立不同组织来源成纤维细胞的培养方法并比较其培养差异.方法 用消化法和贴壁法分离人胚胸主动脉成纤维细胞与人胚肺成纤维细胞,利用差速培养法纯化成纤维细胞,并对所培养的细胞进行倒置显微镜观察和苏木素-伊红染色,观察成纤维细胞形态,并对培养的细胞行波形蛋白免疫染色鉴定.结果 接种24h后人胚胸主动脉成纤维细胞全部贴壁,48h后细胞体积增大并伸出伪足,形状以梭形或不规则形为主;而消化的人胚肺组织中细胞形态不一,经继续培养4~5代后,培养物完全由成纤维细胞构成.结论 用胶原酶I消化人胚胸主动脉成纤维细胞效果好,而用胰蛋白酶液消化人胚肺成纤维细胞效果较好,用差速生长纯化的成纤维细胞可用于实验研究.%Objective To set up the method of the culture of human fibroblasts from different sources and compare their discrepancy. Methods Human embryonic thoracic aortic fibroblasts and embryonic lung fibroblasts were isolated by enzyme digestion and adherence,and purified by means of differential attachment technique. The morphology and phenotype characterization of cultured fibroblasts was inspected by inverted microscope after hematoxylin-eosin staining and the cells were identified by immunocytochemical staining with vimentin. Results The human embryonic thoracic aortic fibroblasts were all adhere to the surface of petri dish after inoculating for 24 hours, volume of the cells enlarged and pseudopodia whose main shapes were clostridial form or irregular stretched out after inoculating for 48 hours. The shapes of human embryonic lung fibroblasts which were isolated by enzyme digestion were differ,and the culture was all composed of fibroblasts after subculturing for 4-5 generations. Conclusion The effect of the method which isolate human embryonic thoracic aortic fibroblasts by collagen I is good. The effect of the method which isolate embryonic

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-04

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

  5. [The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

    Science.gov (United States)

    Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

    2014-01-01

    It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

  6. Macro-micro imaging of cardiac-neural circuits in co-cultures from normal and diseased hearts.

    Science.gov (United States)

    Bub, Gil; Burton, Rebecca-Ann B

    2015-07-15

    The autonomic nervous system plays an important role in the modulation of normal cardiac rhythm, but is also implicated in modulating the heart's susceptibility to re-entrant ventricular and atrial arrhythmias. The mechanisms by which the autonomic nervous system is pro-arrhythmic or anti-arrhythmic is multifaceted and varies for different types of arrhythmia and their cardiac substrates. Despite decades of research in this area, fundamental questions related to how neuron density and spatial organization modulate cardiac wave dynamics remain unanswered. These questions may be ill-posed in intact tissues where the activity of individual cells is often experimentally inaccessible. Development of simplified biological models that would allow us to better understand the influence of neural activation on cardiac activity can be beneficial. This Symposium Review summarizes the development of in vitro cardiomyocyte cell culture models of re-entrant activity, as well as challenges associated with extending these models to include the effects of neural activation.

  7. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  8. Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue.

    Directory of Open Access Journals (Sweden)

    Darryl R Davis

    Full Text Available BACKGROUND: At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs. Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential of CDCs. METHODOLOGY/PRINCIPAL FINDINGS: CPCs were expanded from human endomyocardial biopsies (n = 160, adult bi-transgenic MerCreMer-Z/EG mice (n = 6, adult C57BL/6 mice (n = 18, adult GFP(+ C57BL/6 transgenic mice (n = 3, Yucatan mini pigs (n = 67, adult SCID beige mice (n = 8, and adult Wistar-Kyoto rats (n = 80. Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit(+, endothelial cells (CD31(+, CD34(+, and mesenchymal cells (CD90(+. Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that

  9. 成纤维细胞增殖改变心脏兴奋传导和收缩的仿真研究%Fibroblast proliferation alters cardiac excitation conduction and contraction:a computational study

    Institute of Scientific and Technical Information of China (English)

    He-qing ZHAN; Ling XIA; Guo-fa SHOU; Yun-liang ZANG; Feng LIU; Stuart CROZIER

    2014-01-01

    研究目的:通过建立心肌-成纤维细胞电力耦合模型,探讨成纤维细胞增殖对心脏兴奋传导和力学收缩的影响。  创新要点:基于人心室肌细胞的实验数据,修正了 ten Tusscher等人发表的心室肌模型,构建心肌细胞-成纤维细胞的耦合模型,仿真了成纤维细胞对心肌组织电生理及力学的影响。通过改变耦合模型中重要参数的数值,如网格分辨率、成纤维细胞模型参数、缝隙连接电导等,观察其对心脏去极化、复极化和动作电位周期的影响。  重要结论:在细胞水平上,耦合成纤维细胞使心室肌细胞的动作电位周期延长,主动张力峰值下降(见图4、5)。在组织水平上,成纤维细胞增殖降低兴奋波传导速度并引发传导阻滞(见图6),降低成纤维增殖区域的应变,延长组织的去极化和复极化(见图8),并维持折返(见图10)。%In this study, the effects of cardiac fibroblast proliferation on cardiac electric excitation conduction and mechanical contraction were investigated using a proposed integrated myocardial-fibroblastic electromechanical model. At the cellular level, models of the human ventricular myocyte and fibroblast were modified to incorporate a model of cardiac mechanical contraction and cooperativity mechanisms. cellular electromechanical coupling was realized with a calcium buffer. At the tissue level, electrical excitation conduction was coupled to an elastic mechanics model in which the finite difference method (FDM) was used to solve electrical excitation equations, and the finite element method (FEM) was used to solve mechanics equations. The electromechanical properties of the proposed integrated model were investigated in one or two dimensions under normal and ischemic pathological conditions. Fibroblast proliferation slowed wave propagation, induced a conduction block, decreased strains in the fibroblast proliferous

  10. A simple and effective protocol for fast isolation of human Tenon's fibroblasts from a single trabeculectomy biopsy - a comparison of cell behaviour in different culture media.

    Science.gov (United States)

    Przekora, Agata; Zarnowski, Tomasz; Ginalska, Grazyna

    2017-01-01

    Human Tenon's fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco's modified Eagle's medium (DMEM). We used Eagle's minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM. Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 10(6) vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production. Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some

  11. Therapy of Chronic Cardiosclerosis in WAG Rats Using Cultures of Cardiovascular Cells Enriched with Cardiac Stem Cell.

    Science.gov (United States)

    Chepeleva, E V; Pavlova, S V; Malakhova, A A; Milevskaya, E A; Rusakova, Ya L; Podkhvatilina, N A; Sergeevichev, D S; Pokushalov, E A; Karaskov, A M; Sukhikh, G T; Zakiyan, S M

    2015-11-01

    We developed a protocol for preparing cardiac cell culture from rat heart enriched with regional stem cells based on clonogenic properties and proliferation in culture in a medium with low serum content. Experiments on WAG rats with experimental ischemic myocardial damage showed that implantation of autologous regional stem cells into the left ventricle reduced the volume of cicatricial tissue, promoted angiogenesis in the damaged zone, and prevented the risk of heart failure development.

  12. Lack of cardiac differentiation in c-kit-enriched porcine bone marrow and spleen hematopoietic cell cultures using 5-azacytidine

    NARCIS (Netherlands)

    M.L. Ramirez (Mario); T. McMorrow (Tara); T.M. Sanderson (Thomas M.); C.J. Lancos (C.); Y.-L. Tseng (Y.); D.K.C. Cooper (David); F.J.M.F. Dor (Frank)

    2005-01-01

    textabstractThe adult spleen is a source of early hematopoietic stem cells (HSC). We therefore studied whether culturing spleen or bone marrow (BM) HSC in medium containing 5-azacytidine could induce a cardiac phenotype. c-kit enrichment and depletion of adult pig spleen and BM mononuclear cells wer

  13. Apoptotic markers in cultured fibroblasts correlate with brain metabolites and regional brain volume in antipsychotic-naive first-episode schizophrenia and healthy controls.

    Science.gov (United States)

    Batalla, A; Bargalló, N; Gassó, P; Molina, O; Pareto, D; Mas, S; Roca, J M; Bernardo, M; Lafuente, A; Parellada, E

    2015-08-25

    Cultured fibroblasts from first-episode schizophrenia patients (FES) have shown increased susceptibility to apoptosis, which may be related to glutamate dysfunction and progressive neuroanatomical changes. Here we determine whether apoptotic markers obtained from cultured fibroblasts in FES and controls correlate with changes in brain glutamate and N-acetylaspartate (NAA) and regional brain volumes. Eleven antipsychotic-naive FES and seven age- and gender-matched controls underwent 3-Tesla magnetic resonance imaging scanning. Glutamate plus glutamine (Glx) and NAA levels were measured in the anterior cingulate (AC) and the left thalamus (LT). Hallmarks of apoptotic susceptibility (caspase-3-baseline activity, phosphatidylserine externalization and chromatin condensation) were measured in fibroblast cultures obtained from skin biopsies after inducing apoptosis with staurosporine (STS) at doses of 0.25 and 0.5 μM. Apoptotic biomarkers were correlated to brain metabolites and regional brain volume. FES and controls showed a negative correlation in the AC between Glx levels and percentages of cells with condensed chromatin (CC) after both apoptosis inductions (STS 0.5 μM: r = -0.90; P = 0.001; STS 0.25 μM: r = -0.73; P = 0.003), and between NAA and cells with CC (STS 0.5 μM induction r = -0.76; P = 0.002; STS 0.25 μM r = -0.62; P = 0.01). In addition, we found a negative correlation between percentages of cells with CC and regional brain volume in the right supratemporal cortex and post-central region (STS 0.25 and 0.5 μM; P < 0.05 family-wise error corrected (FWEc)). We reveal for the first time that peripheral markers of apoptotic susceptibility may correlate with brain metabolites, Glx and NAA, and regional brain volume in FES and controls, which is consistent with the neuroprogressive theories around the onset of the schizophrenia illness.

  14. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  15. Optimal primary culture conditions of human gastric mucosal fibroblasts%人胃黏膜成纤维细胞原代培养条件的优化

    Institute of Scientific and Technical Information of China (English)

    张浩; 申晓军; 唐古生; 毕建威

    2009-01-01

    目的 通过优化人胃黏膜成纤维细胞的原代培养条件,提高培养成功率,为进一步研究胃癌相关成纤维细胞奠定基础.方法 原代培养采用植块法,利用免疫组织化学、细胞形态学和电镜等方法进行细胞鉴定;分析不同培养表面、培养基和培养液pH值对原代细胞培养游出情况的影响,筛选适合的培养条件.结果 Logistic逐步回归分析提示RPMI 1640培养基的Waldχ2值=32.4533,P0.05),而且各培养条件之间无交互作用.结论 通过优化培养表面、培养基种类和pH值等培养条件,可以提高人胃黏膜成纤维细胞原代培养的成功率.%Objective To optimize primary culture conditions of human gastric mucosal fibre- blasts for further investigations of gastric cancer associated fibroblasts. Methods The fibroblasts were cul-tured primarily by explant method, and identified by immunohistochemistry, morphology and electron mi-croscopy. The effects of different culture conditions on emigration ratios of ceils were investigated to opti-mize the culture surface, medium and pH in primary culture of gastric mucosal fibroblasts. Results When the other conditions were the same,no significandy different emigration rate was found on the mouse tail-coated surface,fetal bovine serum-coated surface ,and nothing coated surface (P > 0.05 ). As to the role of different culture media including RPMI 1640 medium, DMEM and high-gluco DMEM,there was signifi-cantly higher emigration rate in the RPMI 1640 medium compared with other two media (P < 0.01, stand- ardized estimate = 0. 4688 ). Different pH values(pH = 7.0,7.4 and 7.8)of culture solution led to differ-ent emigration rate,and the best emigration rate was found in pH = 7.4 solution(P < 0.01, standardized estimate =0.5857 ). Furthermore, there was no interaetional effect on the cell emigration rate among cul- ture conditions. Conclusion The primary culture of human gastric mucosal fibroblasts can be greatly im-proved by

  16. Prenatal diagnosis of Zellweger syndrome by measurement of very long chain fatty acid (C26 : 0) β-oxidation in cultured chorionic villous fibroblasts: Implications for early diagnosis of other peroxisomal disorders

    NARCIS (Netherlands)

    Wanders, R.J.A.; Wijland, M.J.A. van; Roermund, C.W.T. van; Schutgens, R.B.H.; Bosch, H. van den; Tager, J.M.; Nijenhuis, A.; Tromp, A.

    1987-01-01

    In this paper we show that cultured chorionic villous fibroblasts efficiently catalyse the peroxisomal β-oxidation of hexacosanoic acid (cerotic acid), a saturated very long chain fatty acid containing 26 carbon atoms. Hexacosanoic β-oxidation was found to be strongly impaired in cultured chorionic

  17. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  18. A Co-Culture Model of Fibroblasts and Adipose Tissue-Derived Stem Cells Reveals New Insights into Impaired Wound Healing After Radiotherapy

    Directory of Open Access Journals (Sweden)

    Frank Haubner

    2015-10-01

    Full Text Available External radiation seems to be associated with increased amounts of cytokines and other cellular modulators. Impaired microcirculation and fibrosis are examples of typical long term damage caused by radiotherapy. Adipose tissue-derived stem cells (ASC are discussed to enhance wound healing, but their role in wounds due to radiotherapy is poorly understood. Normal human fibroblasts (NHF and ASCs were co-cultured and external radiation with doses from 2–12 Gray (Gy was delivered. Cell proliferation and mRNA levels of matrix metalloproteinases (MMP1, MMP2 and MMP13 were determined 48 h after irradiation of the co-cultures by qPCR. Additionally, tissue inhibitors of matrix metalloproteinases (TIMP1, TIMP2 were determined by enzyme-linked immunosorbent assay (ELISA. There was a reduction of cell proliferation after external radiation in mono-cultures of NHFs and ASCs compared to controls without irradiation. The co-culture of ASCs and NHFs showed reduced impairment of cell proliferation after external radiation. Gene expression of MMP1 and MMP13 was reduced after external irradiation in NHF. MMP2 expression of irradiated NHFs was increased. In the co-culture setting, MMP1 and MMP2 gene expression levels were upregulated. TIMP1 and TIMP2 protein expression was increased after irradiation in NHFs and their co-cultures with ASCs. ASCs seem to stimulate cell proliferation of NHFs and modulate relevant soluble mediators as well as proteinases after external radiation.

  19. A Co-Culture Model of Fibroblasts and Adipose Tissue-Derived Stem Cells Reveals New Insights into Impaired Wound Healing After Radiotherapy.

    Science.gov (United States)

    Haubner, Frank; Muschter, Dominique; Pohl, Fabian; Schreml, Stephan; Prantl, Lukas; Gassner, Holger G

    2015-10-29

    External radiation seems to be associated with increased amounts of cytokines and other cellular modulators. Impaired microcirculation and fibrosis are examples of typical long term damage caused by radiotherapy. Adipose tissue-derived stem cells (ASC) are discussed to enhance wound healing, but their role in wounds due to radiotherapy is poorly understood. Normal human fibroblasts (NHF) and ASCs were co-cultured and external radiation with doses from 2-12 Gray (Gy) was delivered. Cell proliferation and mRNA levels of matrix metalloproteinases (MMP1, MMP2 and MMP13) were determined 48 h after irradiation of the co-cultures by qPCR. Additionally, tissue inhibitors of matrix metalloproteinases (TIMP1, TIMP2) were determined by enzyme-linked immunosorbent assay (ELISA). There was a reduction of cell proliferation after external radiation in mono-cultures of NHFs and ASCs compared to controls without irradiation. The co-culture of ASCs and NHFs showed reduced impairment of cell proliferation after external radiation. Gene expression of MMP1 and MMP13 was reduced after external irradiation in NHF. MMP2 expression of irradiated NHFs was increased. In the co-culture setting, MMP1 and MMP2 gene expression levels were upregulated. TIMP1 and TIMP2 protein expression was increased after irradiation in NHFs and their co-cultures with ASCs. ASCs seem to stimulate cell proliferation of NHFs and modulate relevant soluble mediators as well as proteinases after external radiation.

  20. Development of a chemically defined in vitro culture system to effectively stimulate the proliferation of adult human dermal fibroblasts.

    Science.gov (United States)

    Kim, Min Seong; Yun, Jung Im; Gong, Seung Pyo; Ahn, Ji Yeon; Lim, Jeong Mook; Song, Young Han; Park, Kyu Hyun; Lee, Seung Tae

    2015-07-01

    Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 μg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 μg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

  1. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase+catalase and vitamins E and C.

    Science.gov (United States)

    Anane, R; Creppy, E E

    2001-09-01

    Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity and carcinogenicity of many xenobiotics. In the present study, we investigated the possible induction of lipid peroxidation by aluminium in human foreskin fibroblast cultures by assaying the malondialdehyde (MDA) produced inside the cells. The MDA-thiobarbituric acid (TBA) adduct was assayed by HPLC using fluorometric quantification after extraction in n-butanol. Lactate dehydrogenase (LDH) release was used as a marker of aluminium toxicity. MDA production was significantly increased after 24 h incubation with aluminium and paralleled LDH release. Superoxide dismutase (SOD)+catalase and vitamins C and E added in the culture medium as oxygen radical and free radical scavengers were efficient in preventing MDA production by aluminium, indicating that oxidative processes are one of the main pathways whereby this metal induces cytotoxicity. The latter is also largely prevented, thus confirming the link between oxidative stress induced by aluminium and its cytotoxicity in human skin fibroblasts.

  2. Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: absence of a defect in cystinuria and Hartnup disease.

    Science.gov (United States)

    Groth, U; Rosenberg, L E

    1972-08-01

    Transport of lysine, arginine, cystine, and tryptophan was studied in cultured skin fibroblasts from normal controls and from patients with cystinuria and Hartnup disease. Each of these amino acids was accumulated against concentration gradients by energy-dependent, saturable mechanisms. Lysine and arginine were each transported by two distinct processes which they shared with each other and with ornithine. In contrast, cystine was taken up by a different transport system with no demonstrable affinity for the dibasic amino acids. The time course and Michaelis-Menten kinetics of lysine and cystine uptake by cells from three cystinuric patients differed in no way from those found in control cells. Similarly, the characteristics of tryptophan uptake by cells from a child with Hartnup disease were identical to those noted in control cells. These findings indicate that the specific transport defects observed in gut and kidney in cystinuria and Hartnup disease are not expressed in cultured human fibroblasts, thus providing additional evidence of the important role that cellular differentiation plays in the regulation of expression of the human genome.

  3. Uptake of (/sup 3/H)vitamin D/sub 3/ from low and high density lipoproteins by cultured human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shireman, R.B.; Williams, D.; Remsen, J.F.

    1986-03-01

    The plasma distribution and cellular uptake of (/sup 3/H)vitamin D/sub 3/ was studied in vitro using cultured human fibroblasts. Incubation of (/sup 3/H)vitamin D/sub 3/ (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from (/sup 3/H) 1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of (/sup 3/H)vitamin D/sub 3/ from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D/sub 3/ was time, concentration, and temperature dependent. At a concentration of 50 ..mu..g LDL/ml of medium, the uptake of (/sup 3/H)vitamin D/sub 3/ from LDL at 37/sup 0/C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D/sub 3/ absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.

  4. Probing tumor-stroma interactions and response to photodynamic therapy in a 3D pancreatic cancer-fibroblast co-culture model

    Science.gov (United States)

    Glidden, Michael D.; Massodi, Iqbal; Rizvi, Imran; Celli, Jonathan P.; Hasan, Tayyaba

    2012-02-01

    Pancreatic ductal adenocarcinoma is a lethal disease that is often unresectable by the time of diagnosis and is typically non-responsive to chemo- and radiotherapy, resulting in a five year survival of only 3%. Tumors of the pancreas are characterized by a dense fibrous stroma rich in extracellular matrix proteins, which is implicated in poor therapeutic response, though its precise roles remain poorly understood. Indeed, while the use of therapeutics that target the stroma is an emerging paradigm in the clinical management of this disease, the primary focus of such efforts is to enhance drug penetration through dense fibrous stroma and it is unclear to what extent the characteristically rigid stroma of pancreatic tumors imparts drug resistance by acting as a complex signaling partner, or merely as a physical barrier for drug delivery. Here we use 3D in vitro co-cultures of pancreatic cancer cells and normal human fibroblasts as a model system to study heterotypic interactions between these populations. Leveraging this in vitro model along with image-based methods for quantification of growth and therapeutic endpoints, we characterize these co-cultures and examine the role of verteporfin-based photodynamic therapy (PDT) for targeting tumor-fibroblast interactions in pancreatic tumors.

  5. Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

    Science.gov (United States)

    Bressan, F F; Miranda, M S; Bajgelman, M C; Perecin, F; Mesquita, L G; Fantinato-Neto, P; Merighe, G F K; Strauss, B E; Meirelles, F V

    2013-04-01

    Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed

  6. Effects of lower fluence pulsed dye laser irradiation on production of collagen and the mRNA expression of collagen relative gene in cultured fibroblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    YU Hai-yan; CHEN Da-fang; WANG Qi; CHENG Hao

    2006-01-01

    Background Lower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser (585 nm) on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro.Methods Cultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser ( fluence 3 J/cm2,4 J/cm2, spot size 7 mm, pulse duration 450 μs). The production of collagen and the mRNA expression of transforming growth factor (TGF)-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen α1, α2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction.Results The production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm2 (P <0.001). The mRNA expression of TGF- β1,SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm2 (P <0.001). No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm2 (P >0.05).Conclusions Lower fluence (3 J/cm2) pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-β1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.

  7. Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing endogenous avian retrovirus EAV-0 RNA.

    OpenAIRE

    Weissmahr, R N; Schüpbach, J; Böni, J

    1997-01-01

    We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associ...

  8. In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

    Directory of Open Access Journals (Sweden)

    F Di Meglio

    2009-08-01

    Full Text Available The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (a-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively inclines toward an increase in both a-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

  9. Quantitative analysis of cardiac tissue including fibroblasts using three-dimensional confocal microscopy and image reconstruction: towards a basis for electrophysiological modeling

    NARCIS (Netherlands)

    Schwab, Bettina C.; Seemann, Gunnar; Lasher, Richard A.; Torres, Natalia S.; Wülfers, Eike M.; Arp, Maren; Carruth, Eric D.; Bridge, John H.B.; Sachse, Frank B.

    2013-01-01

    Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization bas

  10. Autoantibody against Cardiac β1-Adrenoceptor Induces Apoptosis in Cultured Neonatal Rat Cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yan GAO; Hui-Rong LIU; Rong-Rui ZHAO; Jian-Ming ZHI

    2006-01-01

    To clarify whether apoptosis is involved in the injury processes induced by autoantibody against cardiac β1-adrenoceptor, we investigated the biological and apoptotic effects of antibodies on cultured neonatal rat cardiomyocytes. Wistar rats were immunized with peptides corresponding to the second extracellular loop of the β1-adrenoceptor to induce the production of anti-β1-adrenoceptor antibodies in the sera.Immunoglobulin (Ig) G in the sera was detected using synthetic antigen enzyme-linked immunosorbent assay and purified using the diethylaminoethyl cellulose ion exchange technique. Apoptosis of cardiomyocytes was evaluated using agarose gel electrophoresis and flow cytometry. Our results showed that the positive serum IgG greatly increased the beating rates of cardiomyocytes and showed an "agonist-like" activity. Furthermore, positive serum IgG induced cardiomyocyte apoptosis after treatment with β1adrenoceptor overstimulation for 48 h. The effects of monoclonal antibody against β1-adrenoceptor were also found to be similar to those of positive serum IgG. It was suggested that the autoantibody could induce cardiomyocyte apoptosis by excessive stimulation of β1-adrenoceptor.

  11. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration.

    Science.gov (United States)

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P

    2014-04-01

    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF.

  12. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

    Science.gov (United States)

    Nass, Norbert; Weissenberg, Kristian; Somoza, Veronika; Ruhs, Stefanie; Silber, Rolf-Edgar; Simm, Andreas

    2014-03-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction. Effects of food extracts are often initially analysed in cellular test systems and it is not clear how different cell culture conditions might influence the results. Therefore, we compared the effects of two models for AGE-rich food, bread crust and coffee extract (CE) on WI-38 human lung fibroblasts under different cell culture conditions (sub-confluent versus confluent cells, with and without serum). WI-38 cells responded to coffee and bread crust extract (BCE) with a rapid phosphorylation of PKB (AKT), p42/44 MAPK (ERK 1/2) and p38 MAPK, strongly depending on culture conditions. BCE resulted in increased cell numbers, whereas CE appeared to be cytotoxic. When cell numbers under all culture conditions and treatments were correlated with kinase phosphorylation, the relation between phospho-p38 MAPK and phospho-AKT represented a good, cell culture condition-independent predictor of cell survival.

  13. An Experimental Model Using Cultured Cardiac Myocytes for a Study of the Generation of Premature Ventricular Contractions Under Ultrasound Exposure

    Science.gov (United States)

    Kudo, Nobuki; Yamamoto, Masaya

    2011-09-01

    It is known that use of a contrast agents in echocardiography increases the probability of generation of premature ventricular contractions (PVCs). As a basic study to elucidate the mechanisms and to reduce adverse effects, the generation of PVCs was investigated using cultured cardiac myocytes instead of the intact heart in vivo. Cardiac myocytes were isolated from neonatal rats and cultured on a cover slip. The myocyte sample was exposed to pulsed ultrasound with microbubbles adjacent to the myocytes, and generation of PVCs was examined with ultrasound exposure at various delay times after onset of myocyte contraction. The experimental results showed that generation of PVCs had a stable threshold delay time and that PVCs were generated only when myocytes were exposed to ultrasound with delay times longer than the threshold. The results indicate that the model used in this study is useful for revealing the mechanisms by which PVCs are induced by ultrasound exposure.

  14. Tumor necrosis factor-like weak inducer of apoptosis promotes expression of matrix metalloproteinase 9 in rat cardiac fibroblasts via the nuclear factor-κB pathway%TWEAK通过NF-κB途径促进大鼠成纤维细胞表达基质金属蛋白酶9

    Institute of Scientific and Technical Information of China (English)

    王德金; 任满意; 陈慧娜; 王旭平; 隋树建

    2011-01-01

    目的 研究肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)影响大鼠心肌成纤维细胞(CFs)增殖和胶原合成的作用机制.方法 采用胰酶消化法培养新生Wistar大鼠CFs,加入重组人TWEAK(rhTWEAK)和NF-κB抑制剂PDTC干预,qRT-PCR检测核转录因子NF-κB和金属蛋白酶9(MMP9)mRNA,Western blot检测NF-κB和MMP9蛋白表达,四甲基偶氮唑蓝(MTT)法测定细胞增殖.结果 rhTWEAK作用后NF-κB和MMP9 mRNA表达上调,同时蛋白表达明显增加,PDTC可抑制MMP9蛋白表达和细胞增殖.结论 TWEAK可通过NF-κB途径促进大鼠心肌成纤维细胞表达MMP9.%Objective To investigate the mechanism and effects of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) on the proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs). Methods Cultured CFs from neonatal Wistar rats were isolated by trypsinization and treated with recombinant human TWEAK (rhT-WEAK) and PDTC( NF-kB specific inhibitor). mRNA and protein expressions of NF-kB and MMP9 were detected by qRT-PCR and Western blot, respectively. CFs proliferation was examined by MTT. Results rhTWEAK significantly elevated mRNA and protein expressions of NF-kB and MMP9. PDTC could inhibit MMP9 protein expression and CFs proliferation induced by NF-kB's activation. Conclusion TWEAK can promote expressions of matrix metalloprotein-ase 9 in rat cardiac fibroblasts via the NF-kB pathway.

  15. Alkaline phosphatase expression/activity and multilineage differentiation potential are the differences between fibroblasts and orbital fat-derived stem cells--a study in animal serum-free culture conditions.

    Science.gov (United States)

    Martins, Thaís Maria da Mata; de Paula, Ana Cláudia Chagas; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2014-10-01

    Human orbital fat tissues are a potential source to isolate stem cells for the development of regenerative medicine therapies. For future safe clinical application of these cells, it is critical to establish animal component-free culture conditions as well as to clearly define the stem cell population characteristics differentiating them from other cell types, such as fibroblasts. Therefore, the present study aimed to compare phenotypic and functional characteristics of orbital fat-derived stem cells (OFSCs) and fibroblasts resident in the eyelid skin in donor-matched samples grown in culture medium supplemented with pooled allogeneic human serum (HS) replacing fetal bovine serum (FBS). We first investigated the proliferative effects of OFSCs on HS, and then we compared the alkaline phosphatase (AP) expression and activity, immunophenotypic profile, and in vitro multilineage differentiation potential of OFSCs side-by-side with fibroblasts. The results showed that HS enhanced OFSCs proliferation without compromising their immunophenotype, AP activity, and osteogenic, adipogenic, and chondrogenic differentiation capacities. In contrast to OFSCs, the fibroblasts did not exhibit AP expression and activity and did not have multilineage differentiation potential. The results enabled us to successfully distinguish OFSCs from fibroblasts populations, suggesting that AP expression/activity and multilineage differentiation assays can be used reliably to discriminate mesenchymal stem cells from fibroblasts. Our findings also support the feasibility of pooled allogeneic HS as a safer and more effective alternative to FBS for clinical applications.

  16. Myocardial fibroblast-matrix interactions and potential therapeutic targets.

    Science.gov (United States)

    Goldsmith, Edie C; Bradshaw, Amy D; Zile, Michael R; Spinale, Francis G

    2014-05-01

    The cardiac extracellular matrix (ECM) is a dynamic structure, adapting to physiological and pathological stresses placed on the myocardium. Deposition and organization of the matrix fall under the purview of cardiac fibroblasts. While often overlooked compared to myocytes, fibroblasts play a critical role in maintaining ECM homeostasis under normal conditions and in response to pathological stimuli assume an activated, myofibroblast phenotype associated with excessive collagen accumulation contributing to impaired cardiac function. Complete appreciation of fibroblast function is hampered by the lack of fibroblast-specific reagents and the heterogeneity of fibroblast precursors. This is further complicated by our ability to dissect the role of myofibroblasts versus fibroblasts in myocardial in remodeling. This review highlights critical points in the regulation of collagen deposition by fibroblasts, the current panel of molecular tools used to identify fibroblasts and the role of fibroblast-matrix interactions in fibroblast function and differentiation into the myofibroblast phenotype. The clinical potential of exploiting differences between fibroblasts and myofibroblasts and using them to target specific fibroblast populations is also discussed. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes cultured with high glucose

    Institute of Scientific and Technical Information of China (English)

    LI Xing; LI Mei-rong; GUO Zhi-xin

    2012-01-01

    Background Diabetic cardiomyopathy is the major cause of morbidity and mortality in diabetic patients.Oxidative stress plays an important role in diabetic cardiomyopathy.This study aimed to investigate the effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes (HCM) cultured with high glucose.Methods The cells were assigned to three group: control group,high glucose group and high glucose plus adiponectin group.After culture for 24,48,72 hours,oxidative stress was evaluated by detecting levels of malondialdehyde (MDA)and superoxide dismutase (SOD) in the supernatant of culture media.The expression of p66Shc and Heme oxygenase-1 (HO-1) was detected by real-time polymerase chain reaction (PCR).Flow cytometry was designed to observe and detect cellular apoptosis.Results Our findings showed significant increase in MDA levels and decrease in SOD activity in the high glucose group compared with the control group (P <0.05).However,MDA levels were significantly decreased and SOD activity was significantly increased in the adiponectin group compared with those in the high-glucose group (P <0.05).The mRNA expression of HO-1 in the high glucose group was significantly increased in a time-dependent manner compared with that in the control group (P <0.05).Adiponectin further increased the mRNA expression of HO-1 induced by high glucose in a time-dependent manner (P <0.05).The expression of p66Shc was significantly increased in high glucose group compared with that in the control group (P <0.05).Adiponectin significantly suppressed the upregulation of p66Shc induced by high glucose (P <0.05).The apoptotic rate of cardiomyocytes was significantly increased in the high glucose group compared with that in the control group while the apoptotic rate in the adiponectin group was remarkably declined in comparison with that in the high glucose group.Conclusion Adiponectin reduces high glucose-induced oxidative stress and apoptosis and plays a

  18. Culture of Ear Skin Fibroblasts from the Red Fox (Vulpesvulpes) and Analysis of the Karyotype%赤狐耳成纤维细胞的体外培养及核型分析

    Institute of Scientific and Technical Information of China (English)

    鲍加荣; 王世勇; 郑军军; 杨福合

    2015-01-01

    This study is to isolate and culture skin fibroblasts from the ear of fox.It propels the research on fox somatic nuclear transfer and transgenic fox.In this study,fox ear skin fibroblasts were obtained by tissue pieced culture.The primary culture was digested by trypsin at a short time for the subculture.The fibroblasts were observed by the electron microscope,which had a good growth status.Then karyotype analysis was conduc-ted on the fibroblasts,which showed a standard karyotype of fox.The results showed that fox ear skin fibroblasts could be cultivated successfully in vitro by means of tissues culture method.The primary culture could be puri-fied and ear fibroblast cell line be formed,which can be used as donor cell for nuclear transfer and propels the research on transgenetic special animals.%建立赤狐的耳成纤维细胞的分离和培养体系,为狐的核移植,转基因狐的研究奠定基础.采用组织块培养法分离狐耳成纤维细胞的原代细胞,再利用胰酶短暂消化法进行纯化传代培养,观察成纤维细胞的形态,并进行染色体分析.结果表明,组织块培养法可获得成纤维细胞,经过纯化培养,可获得均一、稳定的狐耳成纤维细胞.可以作为核移植的供体细胞,推动特种动物的转基因研究.

  19. Adsorption of fibronectin and vitronectin onto Primaria and tissue culture polystyrene and relationship to the mechanism of initial attachment of human vein endothelial cells and BHK-21 fibroblasts.

    Science.gov (United States)

    Steele, J G; Dalton, B A; Johnson, G; Underwood, P A

    1995-09-01

    The two cell culture substrata, tissue culture polystyrene (TCPS) and Primaria, were compared in order to determine whether a nitrogen-containing surface such as Primaria attracts cells by a different mechanism to an oxygen-containing surface (TCPS). The amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium onto Primaria and TCPS were determined. Primaria adsorbed two- to threefold more Fn than TCPS, but adsorbed similar amounts of Vn from medium containing FBS. The Fn and Vn binding sites on Primaria were distinct, as adsorption was non-competitive between these two proteins. The amounts of Fn and Vn that adsorbed onto the two surfaces were compared to the concentration dependence of the cell attachment activities of Fn and Vn. Whereas the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment, Primaria adsorbed an Fn surface density that was supraoptimal for attachment of human vein endothelial cells and BHK-21 fibroblasts. We conclude that Primaria differs from TCPS in that both Fn and Vn mediate initial cell attachment to Primaria when the culture medium contains FBS, whereas cell attachment to TCPS is dependent upon Vn.

  20. Focal adhesion kinase activation is required for TNF-α-induced production of matrix metalloproteinase-2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts.

    Science.gov (United States)

    Zhang, Peng; Li, Ya-jing; Guo, Liu-yun; Wang, Guo-fang; Lu, Ke; Yue, Er-li

    2015-08-01

    Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF-α) and its effects on interleukin (IL)-6 and IL-8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP-2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real-time PCR. Tumor necrosis factor alpha dose-dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF-α-induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL-6, IL-8, and MMP-2 in a dose-dependent manner. Knockdown of FAK significantly suppressed TNF-α-induced expression of IL6 and IL8 mRNA and release of IL-6 and IL-8 protein in HPDLFs. Similarly, MMP-2 down-regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF-α-induced IL-6, IL-8, and MMP-2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.

  1. Detection and identification of cancerous murine fibroblasts, transformed by murine sarcoma virus in culture, using Raman spectroscopy and advanced statistical methods.

    Science.gov (United States)

    Salman, A; Shufan, E; Zeiri, L; Huleihel, M

    2013-03-01

    Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer. Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer. In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells. Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80-85% success rate based on their Raman shift spectra. The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites. Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.

  2. Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

    Institute of Scientific and Technical Information of China (English)

    刘剑毅; 李世荣; 纪淑兴

    2004-01-01

    Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P < 0.01 ). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

  3. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile☆☆☆★

    Science.gov (United States)

    Strachan, Alastair J.; Evans, Natalie E.; Williams, O. Martin; Spencer, Robert C.; Greenwood, Rosemary; Probert, Chris J.

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  4. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

  5. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    Science.gov (United States)

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.

  6. Biological responses of human gingival fibroblasts (HGFs in an innovative co-culture model with Streptococcus mitis to thermosets coated with a silver polysaccharide antimicrobial system.

    Directory of Open Access Journals (Sweden)

    Silvia Sancilio

    Full Text Available This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6 and prostaglandin E2 (PGE2 secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE2 production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE2 secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.

  7. Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

    Energy Technology Data Exchange (ETDEWEB)

    Silbert, C.K.; Humphries, D.E.; Palmer, M.E.; Silbert, J.E. (Veterans Administration Outpatient Clinic, Boston, MA (USA))

    1991-02-15

    Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with (3H)glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of (3H)chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

  8. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2012-01-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  9. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2013-02-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  10. In vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and produce a fibrotic-like environment upon stimulation with TGF-β1: Is there a thin line between fetal scarless healing and fibrosis?

    Science.gov (United States)

    Walraven, M; Akershoek, J J; Beelen, R H J; Ulrich, M M W

    2017-03-01

    Transforming growth factor-β (TGF-β) is a cytokine occurring in three isoforms with an important function in development and wound healing. In wound healing, prolonged TGF-β signaling results in myofibroblast differentiation and fibrosis. In contrast, the developing second-trimester fetal skin contains high levels of all three TGF-β isoforms but still has the intrinsic capacity to heal without scarring. Insight into TGF-β signal transduction during fetal wound healing might lead to methods to control the signaling pathway during adult wound healing. In this study, we imitated wound healing in vitro by stimulating fibroblasts with TGF-β1 and examining myofibroblast differentiation. The aim was to gain insight into TGF-β signaling in human fibroblasts from fetal and adult dermis. First, TGF-β1 stimulation resulted in similar or even more severe upregulation of myofibroblast-associated genes in fetal fibroblasts compared to adult fibroblasts. Second, fetal fibroblasts also had higher protein levels of myofibroblast-marker α-smooth muscle actin (α-SMA). Third, stimulated fetal fibroblasts in collagen matrices had higher protein levels of α-SMA, produced more of the fibrotic protein fibronectin splice-variant extra domain A (FnEDA), and showed enhanced contraction. Finally, fetal fibroblasts also produced significant higher levels of TGF-β1. Altogether, these data show that in vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and do produce a fibrotic environment. As healthy fetal skin has high levels of TGF-β1, FnEDA, and collagen-III as well, these findings correlate with the in vivo situation. Therefore, our study demonstrates that there are similarities between fetal skin development and fibrosis and shows the necessity to discriminate between these processes.

  11. Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

    Science.gov (United States)

    Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

    1998-01-01

    Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

  12. 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.

  13. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    Directory of Open Access Journals (Sweden)

    Lindsay M Godin

    Full Text Available The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs and lung fibroblasts (hLFs. Native aged (1 year lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  14. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    Science.gov (United States)

    Godin, Lindsay M; Sandri, Brian J; Wagner, Darcy E; Meyer, Carolyn M; Price, Andrew P; Akinnola, Ifeolu; Weiss, Daniel J; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  15. Increased cholesterol efflux from cultured fibroblasts to plasma from hypertriglyceridemic type 2 diabetic patients: roles of pre beta-HDL, phospholipid transfer protein and cholesterol esterification.

    Science.gov (United States)

    de Vries, R; Groen, A K; Perton, F G; Dallinga-Thie, G M; van Wijland, M J A; Dikkeschei, L D; Wolffenbuttel, B H R; van Tol, A; Dullaart, R P F

    2008-02-01

    We tested whether hypertriglyceridemia associated with type 2 diabetes mellitus is accompanied by alterations in pre beta-HDL, which are considered to be initial acceptors of cell-derived cholesterol, and by changes in the ability of plasma to promote cellular cholesterol efflux. In 28 hypertriglyceridemic and 56 normotriglyceridemic type 2 diabetic patients, and in 56 control subjects, we determined plasma lipids, HDL cholesterol and phospholipids, plasma pre beta-HDL and pre beta-HDL formation, phospholipid transfer protein (PLTP) activity, plasma cholesterol esterification (EST) and cholesteryl ester transfer (CET) and the ability of plasma to stimulate cholesterol efflux out of cultured human fibroblasts. HDL cholesterol and HDL phospholipids were lower, whereas plasma PLTP activity, EST and CET were higher in hypertriglyceridemic diabetic patients than in the other groups. Pre beta-HDL levels and pre beta-HDL formation were unaltered, although the relative amount of pre beta-HDL (expressed as % of total plasma apo A-I) was increased in hypertriglyeridemic diabetic patients. Cellular cholesterol efflux to plasma from hypertriglyceridemic diabetic patients was increased compared to efflux to normotriglyceridemic diabetic and control plasma, but efflux to normotriglyceridemic diabetic and control plasma did not differ. Multiple linear regression analysis demonstrated that cellular cholesterol efflux to plasma was positively and independently related to pre beta-HDL formation, PLTP activity and EST (multiple r=0.48), but not to the diabetic state. In conclusion, cholesterol efflux from fibroblasts to normotriglyceridemic diabetic plasma is unchanged. Efflux to hypertriglyceridemic diabetic plasma is enhanced, in association with increased plasma PLTP activity and cholesterol esterification. Unaltered pre beta-HDL formation in diabetic hypertriglyceridemia, despite low apo A-I, could contribute to maintenance of cholesterol efflux.

  16. Assembly of a functional 3D primary cardiac construct using magnetic levitation

    Directory of Open Access Journals (Sweden)

    Glauco Souza

    2016-07-01

    Full Text Available Easily assembled organotypic co-cultures have long been sought in medical research. In vitro tissue constructs with faithful representation of in vivo tissue characteristics are highly desirable for screening and characteristic assessment of a variety of tissue types. Cardiac tissue analogs are particularly sought after due to the phenotypic degradation and difficulty of culture of primary cardiac myocytes. This study utilized magnetic nanoparticles and primary cardiac myocytes in order to levitate and culture multicellular cardiac aggregates (MCAs. Cells were isolated from 2 day old Sprague Dawley rat hearts and subsequently two groups were incubated with either C1: 33 µL nanoshell/million cells or C2: 50 µL nanoshell/million cells. Varying numbers of cells for each concentration were cultured in a magnetic field in a 24 well plate and observed over a period of 12 days. Constructs generally formed spherical structures. Masson’s trichrome staining of a construct shows the presence of extracellular matrix protein, indicating the presence of functional fibroblasts. Many constructs exhibited noticeable contraction after 4 days of culture and continued contracting noticeably past day 9 of culture. Noticeable contractility indicates the presence of functional primary cardiac myocytes in culture. Phenotypic conservation of cardiac cells was ascertained using IHC staining by α-actinin and collagen. CD31 and fibrinogen were probed in order to assess localization of fibroblasts and endothelial cells. The study verifies a protocol for the use of magnetic levitation in order to rapidly assemble 3D cardiac like tissue with phenotypic and functional stability.

  17. Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia.

    Science.gov (United States)

    Morioka, Norimitsu; Harano, Sakura; Tokuhara, Masato; Idenoshita, Yuko; Zhang, Fang Fang; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-11-01

    The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and (14)C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and (14)C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

  18. Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): comparison with tobacco cigarette smoke extract.

    Science.gov (United States)

    Romagna, Giorgio; Allifranchini, Elena; Bocchietto, Elena; Todeschi, Stefano; Esposito, Mara; Farsalinos, Konstantinos E

    2013-05-01

    Electronic cigarettes (ECs) are used as alternatives to smoking; however, data on their cytotoxic potential are scarce. To evaluate the cytotoxic potential of 21 EC liquids compared to the effects of cigarette smoke (CS). Cytotoxicity was evaluated according to UNI EN ISO 10993-5 standard. By activating an EC device, 200 mg of liquid was evaporated and was extracted in 20 ml of culture medium. CS extract from one cigarette was also produced. The extracts, undiluted (100%) and in five dilutions (50%, 25%, 12.5%, 6.25% and 3.125%), were applied to cultured murine fibroblasts (3T3), and viability was measured after 24-hour incubation by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Viability of less than 70% was considered cytotoxic. CS extract showed cytotoxic effects at extract concentrations above 12.5% (viability: 89.1 ± 3.5% at 3.125%, 77.8 ± 1.8% at 6.25%, 72.8 ± 9.7% at 12.5%, 5.9 ± 0.9% at 25%, 9.4 ± 5.3% at 50% and 5.7 ± 0.7% at 100% extract concentration). Range of fibroblast viability for EC vapor extracts was 88.5-117.8% at 3.125%, 86.4-115.3% at 6.25%, 85.8-111.7% at 12.5%, 78.1-106.2% at 25%, 79.0-103.7% at 50% and 51.0-102.2% at 100% extract concentration. One vapor extract was cytotoxic at 100% extract concentration only (viability: 51.0 ± 2.6%). However, even for that liquid, viability was 795% higher relative to CS extract. This study indicates that EC vapor is significantly less cytotoxic compared tobacco CS. These results should be validated by clinical studies.

  19. Identification of lipid profile on γ-irradiated primary cultured human diploid fibroblasts by UPLC-ESI-QTOF-MS

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Kyeong; Yun, Hyun Jin; Kim, Sun Young; Kim, So Ra; Kim, Eun Ju; Kang, Chang Mo [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Hwang, Geum Sook [Integrated Metabolomics Research Group, Korea Basic Science Institute, Sejong (Korea, Republic of)

    2013-11-15

    The study of biomarkers in normal-tissue radiobiology is of great importance to people subjected to non-destructive testing, to nuclear power industry, and to radiological terrorism. Cytogenetics has remained the 'gold standard' for the detection of chromosomal changes, but this method is cumbersome and time consuming. Therefore, it is necessary to establish a rapid, high-throughput biological monitoring protocol to determine the exposure radiation dose in order to rapidly triage mass casualties. Metabolomics studies is an effective platform for identification and quantification of the complete set of metabolites in a biological system, and it has been successfully provides important insights into physiological and disease states and facilitate in depth understanding of underlying biochemical pathways. Radiation metabolomics has been used to identify radiation effects at the metabolite levels in cells and in urine or serum collected from laboratory animals such as mouse, rat and monkey. Recently, a few radiation metabolomics studies showed that multiple metabolites related to the purine, inositol or pyrimidine metabolism, fatty acid biosynthesis and glycolysis/gluconeogenesis could be potential minimal-invasive markers for radiation exposure. However, there is little consistency in these studies due to differences in analyzed sample (cells, urine, serum), animal models (mouse, rat and monkey). Furthermore, the biological effects of ionizing radiation (IR) were differently regulated in the different absorbed doses, dose-rates and type of IR. Thus, it is necessary to explore the entire biosphere profiling of metabolic markers to make reasonable biological explanation under radiation exposure. We hypothesized that the variations in lipid expression in IR-induced cells would result in a unique lipid profile in human diploid fibroblasts (HDF), which can be detected with the targeted and quantitative metabolomics approach. Irradiated HDF cells showed the dose

  20. The synthetic inhibitor of Fibroblast Growth Factor Receptor PD166866 controls negatively the growth of tumor cells in culture

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    Castelli Mauro

    2009-12-01

    Full Text Available Abstract Background Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1 act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1. Methods Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture. Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay. Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA deriving from the decomposition of poly-unsaturated fatty acids. The expression of Poly-ADP-Ribose-Polymerase (PARP, consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme. Results The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell

  1. Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

    Directory of Open Access Journals (Sweden)

    Stringer Saundra L

    2006-10-01

    Full Text Available Abstract Background Loss of heterozygosity (LOH contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. Results As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. Conclusion Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors

  2. Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

    OpenAIRE

    Bayne, M L; Cascieri, M A; Kelder, B; Applebaum, J; Chicchi, G; Shapiro, J A; Pasleau, F.; Kopchick, J. J.

    1987-01-01

    A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fi...

  3. A Comparison between the Colony Formation of Adult Mouse Spermatogonial Stem Cells in Co cultures with Sertoli and STO (Mouse Embryonic Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Seyed Morteza Koruji

    2010-01-01

    Full Text Available Objective: The aim of this study was to compare the colony formation of spermatogonialstem cells (SSCs on sertoli and STO (Mouse embryonic fibroblast cell line feeder celllayers during a two-week period.Materials and Methods: Initially, sertoli cells and SSCs were isolated from adultmouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristicsof the isolated cells were immunocytochemically confirmed by examiningfor the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF,SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured abovethe Sertoli, STO and the control (without co-culture separately for two weeks. In allthree groups, the number and diameter of colonies were evaluated using an invert microscopeon the 3rd, 7th, 10th and 14th day. β1 and α6-integrin m-RNA expressions wereassessed using a reverse transcription polymerase chain reaction (RT-PCR and realtimePCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR.Statistical analysis was performed using ANOVA; and the paired two-sample t test andTukey’s test were used as post-hoc tests for the data analysis of the three sertoli, STOand control cocultures.Results: At the four specified time points, our results showed significant differences (p<0.05in colony numbers and diameters among the sertoli, STO and control groups. The numberand diameter of colonies increased more rapidly in the sertoli coculture than in the othertwo Our results at all four time points also showed significant differences (p<0.05 in themean colony numbers and diameters between the three groups, with the Sertoli coculturehaving the highest mean values for colony numbers and diameters. The RT-PCR results,after two-weeks of culturing, showed that β1-integrin was expressed in all three groups cocultures,but α6-integrin was not expressed. Additionally, based on real time PCR results,the three genes (β1-integrin, α6-integrin

  4. ICR胎鼠成纤维细胞培养方法的优化%Optimization of the Culture Method of ICR Mouse Embryonic Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    王英; 刘石磊; 刘振伟; 刘云海; 倪和民; 邓桂馨; 郭勇

    2012-01-01

    Objective To investigate a most suitable method for harvesting mouse embryonic fibroblasts ( MEF) ). Methods Three different protocols including tissue only, cell-trypsin digestion, and tissue-trypsin digestion were applied for harvesting and culturing MEF. The MEF growth speed and cell morphology were also compared and analyzed respectively among the 3 methods. Results The cell proliferation was slow, cell size was small, but cells of other types were very few in the tissue only method, but the duration of purification was short. In the cell-trypsin digestion method, the cell proliferation was fast, cell size was somewhat large, but there were a little bit more cells of other types and the duration of purification was somehow longer. Finally, in the tissue-trypsin digestion method, the cell proliferation was very slow, cell size was very small, but there were also very few cells of other types. The duration of purification was very short. Conclusions Our findings indicate that the most efficient method for collecting and culturing mouse embryonic fibroblasts is the protocol of tissue-trypsin digestion.%目的 确定胎鼠成纤维细胞(mouse embryonic fibroblasts,MEF)最优制备方法,为MEF的制备节省时间与原材料.方法 取胎鼠组织,采用组织块培养法、胰酶消化培养法、胰酶消后组织块培养法进行分离培养MEF,比较观察MEF纯度、生长速度、细胞形态等指标,筛选最节省时间与原料的一种培养方法.结果 组织块培养法所得MEF生长速度慢,细胞体积小,杂细胞少,纯化所需传代次数少.胰酶消化培养法的MEF生长速度快,体积较大,杂细胞数量多,纯化所需传代次数较多.胰酶消化后组织块培养法的消化后组织块的成纤维细胞生长速度介于前两种方法之间,细胞体积和形态与组织块法培养结果相似,杂细胞数量较少,纯化所需传代次数少.结论 胰酶消化结合组织块培养法能最大限度的利用材料,在短

  5. The Effect of Walterinnesia aegyptia Venom Proteins on TCA Cycle Activity and Mitochondrial NAD+-Redox State in Cultured Human Fibroblasts

    Directory of Open Access Journals (Sweden)

    Hazem K. Ghneim

    2015-01-01

    Full Text Available Fibroblast cultures were used to study the effects of crude Walterinnesia aegyptia venom and its F1–F7 protein fractions on TCA cycle enzyme activities and mitochondrial NAD-redox state. Confluent cells were incubated with 10 μg of venom proteins for 4 hours at 37°C. The activities of all studied TCA enzymes and the non-TCA mitochondrial NADP+-dependent isocitrate dehydrogenase underwent significant reductions of similar magnitude (50–60% of control activity upon incubation of cells with the crude venom and fractions F4, F5, and F7 and 60–70% for fractions F3 and F6. In addition, the crude and fractions F3–F7 venom proteins caused a drop in mitochondrial NAD+ and NADP+ levels equivalent to around 25% of control values. Whereas the crude and fractions F4, F5, and F7 venom proteins caused similar magnitude drops in NADH and NADPH (around 55% of control levels, fractions F3 and F6 caused a more drastic drop (60–70% of control levels of both reduced coenzymes. Results indicate that the effects of venom proteins could be directed at the mitochondrial level and/or the rates of NAD+ and NADP+ biosynthesis.

  6. Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury.

    Science.gov (United States)

    Jimenez, Ana Gabriela; Harper, James M; Queenborough, Simon A; Williams, Joseph B

    2013-04-15

    A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cellular resistance to cadmium, H(2)O(2), paraquat, thapsigargin, tunicamycium, methane methylsulfonate (MMS) and UV light. Using ANCOVA, we found that the values for the dose that killed 50% of cells (LD(50)) from tropical birds were significantly higher for H(2)O(2) and MMS. When we tested for significance using a generalized least squares approach accounting for phylogenetic relationships among species to model LD(50), we found that cells from tropical birds had greater tolerance for Cd, H(2)O(2), paraquat, tunicamycin and MMS than cells from temperate birds. In contrast, tropical birds showed either lower or no difference in tolerance to thapsigargin and UV light in comparison with temperate birds. These findings are consistent with the idea that natural selection has uniquely fashioned cells of long-lived tropical bird species to be more resistant to forms of oxidative and non-oxidative stress than cells from shorter-lived temperate species.

  7. Exposure of cultured fibroblasts to the peptide PR-11 for the identification of induced proteome alterations and discovery of novel potential ligands.

    Science.gov (United States)

    Breguez, Gustavo Silveira; Neves, Leandro Xavier; Silva, Karina Taciana Santos; de Freitas, Lorran Miranda Andrade; de Oliveira Faria, Gabriela; Isoldi, Mauro César; Castro-Borges, William; de Andrade, Milton Hércules Guerra

    2016-12-01

    The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified molecules were related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-α (HIF-1α) were significantly altered. In addition, at least three differentially expressed molecules of the NF-κB pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the eluted molecules, gC1qR, a known complement receptor, appeared markedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Early cellular responses of BMSCs genetically modified with bFGF/BMP2 co-cultured with ligament fibroblasts in a three-dimensional model in vitro.

    Science.gov (United States)

    Li, Bin; Jha, Ramesh Kumar; Qi, Yong-Jian; Ni, Qu-Bo; Wang, Hui; Chen, Biao; Chen, Liao-Bin

    2016-11-01

    Currently, a number of strategies including the implantation of bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors have been developed to regenerate the tendon-to-bone interface after performing anterior cruciate ligament reconstruction. However, the mechanisms behind the interactions of the implanted BMSCs and tendon cells remain to be elucidated. The aim of this study was to evaluate the early cellular responses of BMSCs genetically modified with basic growth factor growth factor (bFGF)/bone morphogenic protein 2 (BMP2) and ligament fibroblasts in a three-dimensional co-culture model. BMSCs and ligament fibroblasts were both isolated from male Wistar rats. The BMSCs were then transfected with an adenoviral vector carrying bFGF or BMP2. The transfected BMSCs and ligament fibroblasts both encapsulated in alginate beads were co-cultured for 6 days in three-dimensional model. On days 0, 3 and 6, cell proliferation was assayed. On day 6, the expression of several tendon-bone related markers was evaluated. In the co-culture system, bFGF and BMP2 were highly expressed at the mRNA and protein level. During the process, bFGF significantly promoted cell proliferation, as well as the expression of scleraxis (SCX) and collagen (COL) type Ⅰ (COL1) in the BMSCs; however, it markedly decreased the expression of phenotype markers in the ligament fibroblasts, including COL1 and COL3. BMP2 markedly increased the expression of alkaline phosphatase and osteocalcin in the BMSCs and ligament fibroblasts, whereas it had no obvious effect on cell proliferation and collagen synthesis in the ligament fibroblasts. The combination of bFGF and BMP2 resulted in the similarly enhanced proliferation of BMSCs and ligament fibroblasts as observed with bFGF alone; however, this combination more potently promoted osteogenic differentiation than did BMP2 alone. The findings of our study demonstrate the superiority of the combined use of growth factors in inducing

  9. Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs Cultured on an Aligned-Nanofiber Cardiac Patch.

    Directory of Open Access Journals (Sweden)

    Mahmood Khan

    Full Text Available Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.hiPSC-CMs were cultured on; 1 a highly aligned polylactide-co-glycolide (PLGA nanofiber scaffold (~50 microns thick and 2 on a standard flat culture plate. Scanning electron microscopy (SEM was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43 was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic

  10. Effects of TGF-beta and glucocorticoids on map kinase phosphorylation, IL-6/IL-11 secretion and cell proliferation in primary cultures of human lung fibroblasts.

    Science.gov (United States)

    Pelaia, Girolamo; Gallelli, Luca; D'Agostino, Bruno; Vatrella, Alessandro; Cuda, Giovanni; Fratto, Donatella; Renda, Teresa; Galderisi, Umberto; Piegari, Elena; Crimi, Nunzio; Rossi, Francesco; Caputi, Mario; Costanzo, Francesco S; Vancheri, Carlo; Maselli, Rosario; Marsico, Serafino A

    2007-02-01

    Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.

  11. Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

    Science.gov (United States)

    Rogozhnikov, Dmitry; O’Brien, Paul J.; Elahipanah, Sina; Yousaf , Muhammad N.

    2016-01-01

    There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity. PMID:28008983

  12. Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

    Science.gov (United States)

    Rogozhnikov, Dmitry; O’Brien, Paul J.; Elahipanah, Sina; Yousaf, Muhammad N.

    2016-12-01

    There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

  13. Gap Junction Communication and the Propagation of Bystander Effects Induced by Microbeam Irradiation in Human Fibroblast Cultures: The Impact of Radiation Quality

    Science.gov (United States)

    Autsavapromporn, Narongchai; Suzuki, Masao; Funayama, Tomoo; Usami, Noriko; Plante, Ianik; Yokota, Yuichiro; Mutou, Yasuko; Ikeda, Hiroko; Kobayashi, Katsumi; Kobayashi, Yasuhiko; Uchihori, Yukio; Hei, Tom K.; Azzam, Edouard I.; Murakami, Takeshi

    2014-01-01

    Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036–0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from monochromatic 5.35 keV X ray (LET ~6 keV/μm), 18.3 MeV/u carbon ion (LET ~103 keV/μm), 13 MeV/u neon ion (LET ~380 keV/μm) or 11.5 MeV/u argon ion (LET ~1,260 keV/μm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects. PMID:23987132

  14. Fibroblasts in myocardial infarction: a role in inflammation and repair

    Science.gov (United States)

    Shinde, Arti V.; Frangogiannis, Nikolaos G.

    2014-01-01

    Fibroblasts do not only serve as matrix-producing reparative cells, but exhibit a wide range of functions in inflammatory and immune responses, angiogenesis and neoplasia. The adult mammalian myocardium contains abundant fibroblasts enmeshed within the interstitial and perivascular extracellular matrix. The current review manuscript discusses the dynamic phenotypic and functional alterations of cardiac fibroblasts following myocardial infarction. Extensive necrosis of cardiomyocytes in the infarcted heart triggers an intense inflammatory reaction. In the early stages of infarct healing, fibroblasts become pro-inflammatory cells, activating the inflammasome and producing cytokines, chemokines and proteases. Pro-inflammatory cytokines (such as Interleukin-1) delay myofibroblast transformation, until the wound is cleared from dead cells and matrix debris. Resolution of the inflammatory infiltrate is associated with fibroblast migration, proliferation, matrix protein synthesis and myofibroblast conversion. Growth factors and matricellular proteins play an important role in myofibroblast activation during the proliferative phase of healing. Formation of a mature cross-linked scar is associated with clearance of fibroblasts, as poorly-understood inhibitory signals restrain the fibrotic response. However, in the non-infarcted remodeling myocardium, local fibroblasts may remain activated in response to volume and pressure overload and may promote interstitial fibrosis. Considering their abundance, their crucial role in cardiac inflammation and repair, and their involvement in myocardial dysfunction and arrhythmogenesis, cardiac fibroblasts may be key therapeutic targets in cardiac remodeling. PMID:24321195

  15. Trikatu, an herbal compound ameliorates rheumatoid arthritis by the suppression of inflammatory immune responses in rats with adjuvant-induced arthritis and on cultured fibroblast like synoviocytes via the inhibition of the NFκB signaling pathway.

    Science.gov (United States)

    Doss, Hari Madhuri; Ganesan, Ramamoorthi; Rasool, Mahaboobkhan

    2016-10-25

    The present study was designed to investigate the potential therapeutic effect of trikatu, an herbal compound and its underlying molecular mechanism in rats with adjuvant-induced arthritis (AIA). Our results indicate that trikatu (1000 mg/kg/b.wt. oral) administration suppressed the production of pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein (MCP)-1) and downregulated the mRNA expression levels of inflammatory mediators (TNF-α, IL-1β, IL-6, IL-17, MCP-1, receptor activator of nuclear factor kappa B ligand (RANKL), cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS)) and transcription factors (nuclear factor kappa B 65 (NFкB-p65) and activator protein-1 (AP-1)) in cultured AIA-fibroblast like synoviocytes and synovial tissue of AIA rats. Consistently, the protein expression of NFкB-p65, IL-17, TNF-α, COX-2, and RANKL was also dramatically reduced in cultured AIA-fibroblast like synoviocytes and synovial tissue of AIA rats by trikatu treatment. In addition, trikatu suppressed the expression and phosphorylation of NFкB-p65 similar to the Bay 11-7082 (NFкB inhibitor) in cultured AIA-fibroblast like synoviocytes. Furthermore, trikatu alleviated the histopathology of joint of arthritic rats. Overall, these data highlights that trikatu could be a promising alternative modality for the possible treatment of rheumatoid arthritis and other inflammatory diseases.

  16. 氯化胆碱对异丙肾上腺素诱导大鼠心肌成纤维细胞增殖及胶原合成的影响%Effect of choline chloride on isoproterenol-induced proliferation and collagen synthesis of rat cardiac fibroblasts

    Institute of Scientific and Technical Information of China (English)

    范凯; 王超; 宋浩鑫; 蒋雅楠; 刘艳

    2013-01-01

    Objective To investigate the effect of choline chloride (choline) on isoproterenol (Iso)-induced cardiac fibroblasts (CFs) proliferation and collagen synthesis. Methods CFs were isolated from neonatal Wistar rats and cultured in vitro. They were divided into groups as follows: control group, Iso group, Iso + choline group. After 48 h, the proliferation of CFs was measured by MTT assay, the collagen synthesis was detected by hydroxyproline method. Re-sults CFs significantly increased by 10-4 mol/L Iso(P <0. 05) ,1 mmol/L choline obviously inhibited CFs proliferation and collagen synthesis ( P < 0. 05 ) , which were mediated by 10-4 mol/L Iso. Conclusion Choline chloride inhibits the proliferation and collagen synthesis of CFs induced by Iso.%目的 探讨氯化胆碱对异丙肾上腺素(Iso)诱导的心肌成纤维细胞(CFs)增殖和胶原合成的影响.方法 体外培养新生Wistar大鼠心肌成纤维细胞,分为对照组、Iso组和Iso+氯化胆碱组.各组药物分别作用48 h.应用MTF实验方法检测心肌成纤维细胞增殖,羟脯氨酸试剂盒检测胶原合成.结果 10-4 mol/L Iso明显促进CFs增殖(P<0.05),1 mmol/L氯化胆碱明显抑制10-4 mol/L Iso诱导的成纤维细胞增殖(P<0.05)与胶原合成(P<0.05).结论 氯化胆碱抑制异丙肾上腺素诱导的心肌成纤维细胞增殖及胶原合成.

  17. Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

    Science.gov (United States)

    Hashimoto, Shuichi; Nakano, Hiroshi; Suguta, Yuko; Irie, Seiko; Jianhua, Luo; Katyal, Sikardar L.

    2012-01-01

    Objectives Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh(sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies...

  19. Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.

    Directory of Open Access Journals (Sweden)

    Zheng Lao

    Full Text Available Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF spheroids in only 2-3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids. Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

  20. Cytotoxic Evaluation of Elastomeric Dental Impression Materials on a Permanent Mouse Cell Line and on a Primary Human Gingival Fibroblast Culture

    Directory of Open Access Journals (Sweden)

    Roberta Tiozzo

    2009-08-01

    Full Text Available The need for clinically relevant in vitro tests of dental materials is widely recognized. Nearly all dental impression materials are introduced into the mouth just after mixing and allowed to set in contact with the oral tissues. Under these conditions, the materials may be toxic to cells or may sensitize the tissues. The aim of the present study is to evaluate the potential cytotoxicity of new preparations of elastomeric dental impression materials: A four vinylpolysiloxanes: Elite H-D Putty and Elite H-D Light Body (Zhermack, Badia Polesine, Rovigo, Italy; Express Putty and Express Light Body (3M ESPE AG Seefeld, Germany and B two polyethers: Impregum Penta and Permadyne Penta L (3M ESPE AG Seefeld, Germany. The cytotoxicity of these impression materials were examined using two different cell lines: Balb/c 3T3 (permanent cell line and human gingival fibroblasts (primary cell line and their effects were studied by indirect and direct tests. The direct tests are performed by placing one sample of the impression materials in the centre of the Petri dishes at the time of the seeding of cells. The cell growth was evaluated at the 12th and 24th hours by cell number. The indirect tests were performed by incubating a square of 1 cm diameter impression material in 5 mL of medium at 37 °C for 24 hours (“eluates”. Subconfluent cultures are incubated with “eluates” for 24 hours. The MTT-formazan production is the method used for measuring the cell viability. The results indicate that: a polyether materials are cytotoxic under both experimental conditions; b among vinylpolysiloxanes, only Express Light Body (3M ESPE AG Seefeld, Germany induces clear inhibition of cellular viability of Balb/c 3T3 evaluated by direct and indirect tests and c the primary cell line is less sensitive to the toxic effect than the permanent cell line.

  1. Primary Isolation and Culture of Muscovy Duck Embryo Fibroblasts%番鸭胚成纤维细胞的原代分离与培养研究

    Institute of Scientific and Technical Information of China (English)

    沈学怀; 张丹俊; 潘孝成; 戴银; 赵瑞宏; 胡晓苗; 侯红艳; 周学利; 朱传民

    2015-01-01

    In order to establish the primary culture,subculture and cryopreservation techniques of muscovy duck embryo fibroblasts (MDEF),11 day-age muscovy duck embryoes were selected,2.5 g/L trypsin di-gestion of the embryoes and primary MDEF were cultured in DMEM containing 100 mL/L serum.The effects of hot and cold digestion methods on MDEF density and viability were studied.The effects of dif-ferent serum concentrations on MDEF growth and cryopreserved cell viability,and the effects of centrifu-gation on MDEF viability in subculture and resuscitation were investigated.The results showed that this methods could be used for isolation and pure culture of MDEF in vitro .The density and viability of MDEF by cold digestion were higher than the hot digestion.The cells grew faster with the higher serum concen-tration range of 20 mL/L-100 mL/L.Serum concentrations significantly affected MDEF recovery viability;DMSO∶serum∶medium ratio of 1 ∶2∶7 was good for MDEF frozen liquid.It may simplify operations and increase a certain cell viability if the centrifugation in MDEF subculture and recovery process was re-moved.This study established primary isolation and culture methods of MDEF,and laid the foundation for in vitro cell culture system suitable for study of MDEF.%为建立番鸭胚成纤维细胞(MDEF)的体外培养、传代和冻存技术,选用11胚龄番鸭胚,2.5 g/L 胰蛋白酶消化,原代 MDEF 于含100 mL/L 血清的 DMED 培养液中培养;研究热、冷两种消化方法对MDEF 消化密度及活率的影响,探讨不同血清浓度对 MDEF 生长、冻存活性的影响,以及离心对 MDEF 传代和复苏时细胞活率的影响。结果显示,胰酶消化法获得了 MDEF,细胞贴壁紧密,生长良好;冷消化得到的原代 MDEF 密度和活率均高于热消化;血清浓度在20 mL/L~100 mL/L 范围内,浓度越高 MDEF 生长速度越快;血清浓度显著影响 MDEF 复苏的活率;MDEF 冻存液中 DMSO

  2. Respiratory activity and growth of human skin derma fibroblasts.

    Science.gov (United States)

    Papa, F; Scacco, S; Vergari, R; Bucaria, V; Dioguardi, D; Papa, S

    1998-09-01

    A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

  3. Musical rhythms in heart period dynamics: a cross-cultural and interdisciplinary approach to cardiac rhythms.

    Science.gov (United States)

    Bettermann, H; Amponsah, D; Cysarz, D; van Leeuwen, P

    1999-11-01

    The purpose of this study was to expand classic heart period analysis methods by techniques from ethnomusicology that explicitly take complex musical rhythm principles into consideration. The methods used are based on the theory of African music, the theory of symbolic dynamics, and combinatorial theory. Heart period tachograms from 192 24-h electrocardiograms of 96 healthy subjects were transformed into binary symbol sequences that were interpretable as elementary rhythmic (percussive) patterns, the time lines in African music. Using a hierarchical rhythm pattern scheme closely related to the Derler Rhythm Classification (from jazz theory), we calculated the predominance and stability of pattern classes. The results show that during sleep certain classes, specific to individuals, occurred in a cyclically recurrent manner and many times more often than expected. Simultaneously, other classes disappeared more or less completely. Moreover, the most frequent classes obviously originate from phase-locking processes in autonomic regulation (e.g., between respiratory and cardiac cycles). In conclusion, the new interdisciplinary method presented here demonstrates that heart period patterns, in particular those occurring during night sleep, can be interpreted as musical rhythms. This method may be of great potential use in music therapy research.

  4. Fatalism and short-termism as cultural barriers to cardiac rehabilitation among underprivileged men.

    Science.gov (United States)

    Savage, Mathieu; Dumas, Alex; Stuart, Stephen A

    2013-11-01

    Cardiovascular diseases are a leading cause of death and disability in Canada, and individuals of low socioeconomic status appear particularly vulnerable to such disorders. Although many health-related institutions have promoted cardiovascular health and have created cardiac rehabilitation programmes, they have not attained their desired outcomes, especially amongst socioeconomically deprived men. Drawing on Pierre Bourdieu's sociocultural theory, this qualitative study aims to understand the social mechanisms underpinning the lifestyles and health practices of men who had suffered a cardiovascular incident requiring hospitalisation. In all, 20 interviews were conducted with francophone men aged 40 to 65 years living in the province of Québec, Canada. The analysis strongly suggests that the respondents' living conditions and disease were significant obstacles to their adoption of a healthy lifestyle. Their despair and pessimism, apparently originating in the harshness of their financial realities, physical limitations and social networks, led them to believe that they could do little to control their lives, thereby limiting the fulfillment of any long-term ambitions. Therefore, the adoption of a habitus characterised by fatalistic and short-term perceptions of health influenced their lifestyle choices, leading them to maintain lives that were in stark contrast with the recommendations made by health promotion experts.

  5. Protective effects of APP 17-mer peptide on cultured human skin fibroblasts after irradiation with ultraviolet light%APP17肽通过抑制细胞内ROS保护紫外线照射后人皮肤成纤维细胞

    Institute of Scientific and Technical Information of China (English)

    陈慧; 连石; 朱威

    2011-01-01

    Objective Ultraviolet light (UV) is known to cause photoaging of skin.UV irradiation can damage proliferation capacity and induce collagenase in fibroblasts in the dermis .Many researchers have explored the potential photo-protective agents;however,no ideal agent has been widely accepted .Amyloid precursor protein 17-mer peptide (APP17-mer peptide),an active peptide segment,has been reported to be responsible for the trophic effect in clonal CNS neuronal line ,fibroblast cell line and HaCat cells.The aim of this study was to explore the effects of APP17-mer peptide on cultured fibroblasts after ultraviolet irradiation .Methods Human skin fibroblasts were cultured in DMEM medium with or without APP 17-mer peptide (concentrations ranging from 20 μmol/L,40 μmol/L,to 80 μmol/L).The cultured fibroblasts were exposed to a single UV irradiation,and the proliferation activity of fibroblasts was detected by a MTT assay .The ex-pression of matrix metalloproteinase-1 (MMP-1) mRNA was analyzed quantitatively following real -time RT-PCR.The generation of intracellular reactive oxygen species (ROS) was measured with fluorescent quantita-tion method.Results A single exposure to UV irradiation depressed proliferation activity of fibroblasts com -pared with sham-irradiated control (P <0.05).40 μmol/L and 80 μmol/L APP17-mer peptide increased the cellular proliferation activity in UV irradiated and unirradiated fibroblasts (P <0.05),however,20 μmol/L did not show such protective effects (P >0.05).A single exposure of fibroblasts to UV irradiation resulted in 1.78 fold up-regulation of MMP-1 mRNA compared with unirradiated sample (P <0.05),and 40 μmol/L and 80 μmol/L APP17-mer peptide decreased the expression of MMP -1 mRNA (P <0.05 and P <0.01,re-spectively).UV irradiation increased generation of ROS in cultured fibroblasts (P <0.05).40 μmol/L APP17-mer peptide inhibited the generation of ROS in irradiated fibroblasts .Conclusions APP17-mer pep-tide can

  6. 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...... displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology...

  7. Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

    Science.gov (United States)

    Tahara, H; Hara, E; Tsuyama, N; Oda, K; Ide, T

    1994-03-30

    Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

  8. Synergistic anti-inflammatory activity of the antimicrobial peptides human beta-defensin-3 (hBD-3 and cathelicidin (LL-37 in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.

    Directory of Open Access Journals (Sweden)

    Telma Blanca Lombardo Bedran

    Full Text Available Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3 and cathelicidin (LL-37 and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 µM and LL-37 (0.1 and 0.2 µM individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.

  9. Discovery and progress of direct cardiac reprogramming.

    Science.gov (United States)

    Kojima, Hidenori; Ieda, Masaki

    2017-02-14

    Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

  10. Differentiation induction of mouse cardiac stem cells into sinus node-like cells by co-culturing with sinus node.

    Science.gov (United States)

    Fang, Yi-Bing; Liu, Xuan; Wen, Jing; Tang, Xiao-Jun; Yu, Feng-Xu; Deng, Ming-Bin; Wu, Chang-Xue; Liao, Bin

    2014-01-01

    Sinus nodal cells can generate a diastolic or "pacemaker" depolarization at the end of an action potential driving the membrane potential slowly up to the threshold for firing the next action potential. It has been proved that adult cardiac stem cells (CSCs) can differentiate into sinus nodal cells by demethylating agent. However, there is no report about adult CSCs-derived sinus nodal cells with pacemaker current (the funny current, I f). In this study, we isolated the mouse adult CSCs from mouse hearts by the method of tissue explants adherence. The expression of c-kit protein indicated the isolation of CSCs. Then we co-cultured mouse CSCs with mouse sinus node tissue to induce the differentiation of these CSCs into sinus node-like cells, which was proved by identifying the enhanced expression of marker proteins cTnI, cTnT and α-Actinin with Immunofluorescence staining. At the same time, with whole-cell patch-clamp we detected the I f current, which can be blocked by CsCl, in these differentiated cells. In conclusion, by confirming specific I f current in the induced node-like cells, our work shows a method inducing differentiation of CSCs into sinus node-like cells, which can provide helpful information for the further research on sick sinus syndrome.

  11. 脉冲掺钕钇铝石榴石激光对增生性瘢痕成纤维细胞胶原合成的影响%Effects of pulsed Nd:YAG laser on collagen synthesis in hypertrophic scar derived fibroblast cultures

    Institute of Scientific and Technical Information of China (English)

    舒彬; 郝林林; 曾登芬

    2002-01-01

    Objective In order to explore the selectively inhibitory effects of pulsed Nd:YAG laser irradiation on collagen production of scar fibroblasts in vitro.Methods Cultured fibroblasts derived from hypertrophic scars(HS) and normal human skin were irradiated with a pulsed Nd:YAG laser(wavelength 1 064 nm,pulse width 150 μ s) at various energy density levels (500,1 000,1 500 and 2 000 J/cm2).At 24 hours after laser irradiation, collagen production of fibroblasts was measured by the incorporation of 3H proline. The expression of proα 1(I)procollagen mRNA was investigated by blot hybridization techniques. Results Collagen production of HS fibroblasts was significantly increased, as 2 times as that of normal skin fibroblasts. Type I procollagen mRNA level in HS fibroblasts was markedly elevated, as 3 times as that in normal skin fibroblasts.Conclusion Pulsed Nd:YAG laser at energy density of 1 000 J/cm2 can selectively suppress collagen synthesis and type I procollagen mRNA level of HS fibroblasts.

  12. microRNA-21 promotes cardiac fibrosis and development of heart failure with preserved left ventricular ejection fraction by up-regulating Bcl-2.

    Science.gov (United States)

    Dong, Shuguang; Ma, Wenhan; Hao, Bohan; Hu, Fen; Yan, Lianhua; Yan, Xiaofei; Wang, Ya; Chen, Zhijian; Wang, Zhaohui

    2014-01-01

    The morbidity and mortality of heart failure with preserved left ventricular ejection fraction (HFpEF) were similar to those of systolic heart failure, but the pathogenesis of HFpEF remains poorly understood. It was demonstrated that, in systolic heart failure, microRNA-21 (miR-21) could inhibit the apoptosis of cardiac fibroblasts, leading to cardiac hypertrophy and myocardial fibrosis, but the role of miR-21 in HFpEF remains unknown. By employing cell culture technique, rat myocardiocytes and cardiac fibroblasts were obtained. The expression of miR-21 in the two cell types under different conditions was compared and we found that the miR-21 expression was significantly higher in cardiac fibroblasts than in myocardiocytes. We established a rat HFpEF model and harvested the tissues of cardiac apex for pathological examination, Northern blotting and so forth. We found that miR-21 expression was significantly higher in model rats than in sham-operated rats, and the model rats developed the cardiac atrophy and cardiac fibrosis. After injection of miR-21 antagonist, the the cardiac atrophy and cardiac fibrosis were conspicuously ameliorated. Both in vivo and in vitro, inhibition of miR-21 expression resulted in reduced Bcl-2 expression while over-expression of miR-21 led to elevation of Bcl-2 expression. Our study suggested that miR-21 promoted the development of HFpEF by up-regulating the expression of anti-apoptotic gene Bcl-2 and thereby suppressing the apoptosis of cardiac fibrosis.

  13. Effect of TGF-β1 on the expression of collagen in rat atrial and ventricular fibroblasts

    Directory of Open Access Journals (Sweden)

    Fa-jin LIU

    2015-07-01

    Full Text Available Objective To observe the effect of transforming growth factor beta 1 (TGF-β1 on the expression of collagen in rat atrial and ventricular fibroblasts, and to investigate its specific molecular mechanisms. Methods Tissue explant attachment was used to culture fibroblasts obtained from the atrium and ventricle of rat heart, and they were identified with SABC immunocytochemical staining, and then the following experiments were carried out. (1 Hydroxyproline digestion was performed to study the effects of TGF-β1, within different concentrations (0, 5, 10ng/ml and different action time (6, 12, 24, 48h on the content of hydroxyproline in rat's atrial and ventricular fibroblasts. (2 Rat's atrial and ventricular fibroblasts were stimulated with TGF-β1 in optimal concentration and action time, the expression of α-smooth muscle actin (α-SMA was determined with Western blotting, and the expressions of typeⅠand Ⅲ collagen mRNA were evaluated with reverse-transcription PCR. The contents of hydroxyproline in the respective cells were measured with hydroxyproline determination. Western blotting was used to measure the protein expression of Smad2/3, p-Smad2/3 and Smad7. Results (1 TGF-β1 was shown to stimulate the collagen synthesis in rat's atrial and ventricular fibroblasts, and the optimal stimulus was TGF-β1 concentration 5ng/ml with action time of 24h. (2 After being stimulated by optimal stimulation effect of TGF-β1, the expression of typeⅠand Ⅲ collagen and p-Smad2/3 increased,while that of Smad7 decreased significantly only in atrial fibroblasts (P<0.05, but not in ventricular fibroblasts. No statistical difference was found in the expression of Smad2/3 between the atrial and ventricular fibroblasts after being stimulated by TGF-β1 under optimal stimulating conditions. Conclusion TGF-β1 can induce dysbolism of collagen of cardiac fibroblasts with abnormal expression of cytoskeletal protein, which may occur more obviously in rat

  14. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  15. 生脉饮对TNF-α诱导的大鼠心肌成纤维细胞胶原合成的影响%Effect of Shengmai Oral Liquid on TNF-α-induced collagen synthesis in cardiac fibroblasts for rats

    Institute of Scientific and Technical Information of China (English)

    王敬春; 徐波; 张春

    2012-01-01

    目的 探讨生脉饮对肿瘤坏死因子(tumor necrosis factor α,TNF-α)诱导的新生大鼠心肌成纤维细胞(cardiac fibroblasts,CFs)胶原合成的影响及其机制.方法 用不同浓度生脉饮载药血清与TNF-α处理体外培养的CFs,采用分光光度法检测细胞培养上清液中羟脯氨酸含量,采用实时定量RT-PCR检测MMP1和TIMP1 mRNA的表达水平.结果 TNF-α处理组羟脯氨酸含量较空白对照组明显升高;与TNF-α组比较,生脉饮口服液血清中羟脯氨酸含量降低.与空白对照组比较,TNF-α组MMP1和TIMP1 mRNA表达上调;生脉饮载药血清组MMP1较TNF-α组显著升高,而TIPM1显著降低,差异有统计学意义(均P < 0.05),并呈浓度依赖性.结论 生脉饮对TNF-α诱导的CFs 胶原合成具有抑制作用,下调MMP1和上调TIPM1 mRNA表达可能是其作用的机制之一.%Objective To explore the effect and mechanism of Shengmai Oral Liquid on collagen synthesis of neonatal rat cardiac fibroblasts (CFs) induced by tumor necrosis factor (tumor necrosis factor α, TNF-α). Methods Cardiac fibroblasts (CFs) were treated with different concentrations of Shengmai Oral Liquid drug-loaded serum plus TNF-α treatment in vitro. Hydroxyproline content, MMP1 mRNA and TIMP1 mRNA expression were assayed by spectrophotometry and real-time quantitative reverse transcription-PCR, respectively. Results The content of hydroxyproline in cardiac fibroblasts treated with TNF-α was higher than the control group. The content of hydroxyproline was reduced in Shengmai Oral Liquid drug-loaded serum group compared with the TNF-α group. MMP1 and TIMP1 mRNA expression was up-regulated expression in TNF-α groups compared with the control group. MMP1 mRNA expression was up-regulated and TIMP1 was down-regulated in Shengmai Oral Liquid drug-loaded serum group compared with the TNF-α group. Conclusion The results reveal that Shengmai Oral Liquid can inhibit the collagen synthesis in cardiac fibroblasts, and may

  16. Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

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    Aroem Naruni

    2016-01-01

    Full Text Available AbstrakLatar belakang: Lingkungan mikro yaitu sel stromal dam matriks ekstraseluler saat ini dinyatakansebagai kontributor dalam perkembangan tumor. Beberapa penelitian telah mengembangkan matriksekstraseluler yang mendukung perkembangan sel in vitro. Matriks ekstraseluler adalah suatu komplekssusunan supramolekuler dari berbagai macam glycoprotein dan proteoglycan. Matriks ekstraselulermenyediakan integritas jaringan, bertindak sebagai scaffold alami tempat sel melekat dan berinteraksiserta berperan sebagai reservoir pertumbuhan sel. Penelitian ini bertujuan untuk mendapatkan deposisidan deselularisasi yang optimal pada matriks ekstraseluler.Metode: Dalam penelitian ini, kami mengembangkan cells crowder untuk meningkatkan deposit matriksekstraseluler dari kultur sel primer fibroblast payudara yang diperoleh dari spesimen hasil operasimammoplasty. Dextran 500 kDa ditambahkan dalam media kultur DMEM lengkap yang telah ditambahkan0.5% FBS dan 100μM L-ascorbic acid 2-phosphate. Setelah tujuh hari, sel dilisis dengan menggunakanSodium Deoxycolate (DOC.Hasil: Deposisi matriks ekstraseluler dan proses deselulerisasi dari sel primer fibroblas payudara dapatterdeteksi dengan menggunakan antibodi Rabbit anti human fibronectin yang selanjutnya ditambahkandengan anti rabbit IgG yang telah dikonjugasi dengan Alexa Fluor 488.Kesimpulan: Penambahan dextran sulfat dan prosesing lysis dengan sodium deoxycolate dapatmeningkatkan deposisi dan menghasilkan deselularisasi matriks ekstraseluler. (Health Science Journalof Indonesia 2015;6:43-7Kata kunci: matriks ekstra selular, kanker mammae, stem cell, sel fibroblast AbstractBackground: The microenvironment including stromal cells and extracellular matrix (ECM is now consideredan active contributor to tumor progression. Certain studies have developed ECM which supports a suitable cellulargrowth in vitro. The ECM is a complex supramolecular assembly of a variety of glycoproteins and proteoglycans

  17. Cholesteatoma fibroblasts promote epithelial cell proliferation through overexpression of epiregulin.

    Directory of Open Access Journals (Sweden)

    Mamoru Yoshikawa

    Full Text Available To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b. To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial-mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.

  18. Scanning electron microscopic, transmission electron microscopic, and confocal laser scanning microscopic observation of fibroblasts cultured on microgrooved surfaces of bulk titanium substrata

    NARCIS (Netherlands)

    den Braber, E.T.; Jansen, Henricus V.; de Boer, Meint J.; Croes, H.J.E.; Elwenspoek, Michael Curt; Ginsel, L.A.; Jansen, J.A.

    1998-01-01

    During this study, microtechnology and plasma etching were used to produce gratings 1.0 (TiD01), 2.0 (TiD02), 5.0 (TiD05), and 10.0 µm wide (TiD10) into commercially pure titanium wafers. After incubation of rat dermal fibroblast (RDFs) on these surfaces for 3 days, the cells were observed with

  19. Induction of matrix metalloproteinase-1 by tumor necrosis factor-α is mediated by interleukin-6 in cultured fibroblasts of keratoconus.

    Science.gov (United States)

    Du, Genlai; Liu, Chengxing; Li, Xiaona; Chen, Weiyi; He, Rui; Wang, Xiaojun; Feng, Pengfei; Lan, Weiwei

    2016-12-01

    Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.

  20. Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

    Science.gov (United States)

    Hendudari, Farzane; Piryaei, Abbas; Hassani, Seyedeh-Nafiseh; Darbandi, Hasan; Bayat, Mohammad

    2016-05-01

    Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.

  1. 汉防己甲素诱导人眼Tenon囊成纤维细胞的凋亡效应%Inducement effect of tetrandrine on the apoptosis of cultured human Tenon's capsule fibroblasts

    Institute of Scientific and Technical Information of China (English)

    李岱; 杨柳; 林少春; 吴开力

    2010-01-01

    目的 研究汉防己甲素(Tet)抑制人眼Tenon囊成纤维细胞(TCFs)的凋亡效应.方法 用组织块混合消化液培养法体外培养人眼TCFs,并对培养的传代细胞进行形态学鉴定.通过透射电镜、TUNEL法和流式细胞仪观察Tet抑制人眼TCFs的凋亡作用.结果 人眼TCFs体外生长良好.透射电镜下见Tet处理后的人眼TCFs细胞质固缩,呈现凋亡表现;TUNEL法可见Tet组出现大量的阳性凋亡细胞;流式细胞仪检测TCFs经Tet作用后G_0/G_1期细胞减少22.2%,S期细胞增加20.53%,G_2/M期细胞增加1.6%,Tet抑制TCFs细胞周期于G1期,Tet组凋亡率明显高于对照组(P=0.000).结论 Tet可以诱导人眼TCFs发生凋亡,进而发挥其抑制增生的作用.%BackgroundExcessive conjunctival scar formation is a main cause of filtering surgical failure in glaucoma.It has been reported that the failure rate of filtering surgery is a tough problem in the research of glaucoma.Research showed that tetrandrine (Tet) suppress the proliferation of cultured human fibroblasts of Tenons capsule (TCFs) in vitro,but its possible mechanism is still unclear up to now.ObjectiveThe aim of this study is to investigate the inducement effects of Tet on the apoptosis of fibroblasts of Tenons capsule and mechanism.MethodsThe Tenon's capsule was obtained from donor eyes of Zhongshan Eye Center Eye Bank for further use.Human Tenon's capsule fibroblasts were cultured by explant culture method in mixed medium in vitro and the third to seventh generations of cells were collected for the experiment.The subcultured cells were identified by morphology observation and Vimentin staining.The apoptosis of cultured fibroblasts in Tet-treated group and control group was studied by using TUNEL (TdT- mediated dUTP nick end labling,TUNEL),flow cytometry (FCM) and transmission electron microscope.The cell number in different cellular cycle was calculated in Ted-treated group and control group.Results The cultured cells reached

  2. The Role of Cardiac Side Population Cells in Cardiac Regeneration

    Science.gov (United States)

    Yellamilli, Amritha; van Berlo, Jop H.

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies. PMID:27679798

  3. [Infective endocarditis in intensive cardiac care unit - clinical and biochemical differences of blood-culture negative infective endocarditis].

    Science.gov (United States)

    Kaziród-Wolski, Karol; Sielski, Janusz; Ciuraszkiewicz, Katarzyna

    2017-01-23

    Diagnosis and treatment of infective endocarditis (IE) is still a challenge for physicians. Group of patients with the worst prognosis is treated in Intensive Cardiac Care Unit (ICCU). Etiologic agent can not be identified in a substantial number of patients.

  4. Study on Isolation, Passage, Cryopreservation and Histology of Mouse Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    ZHANG Gui-xue; LIU Yan; HU Peng-fei

    2004-01-01

    The embryonic ages were determined for the best preparation of mouse fibroblasts. Four methods were adapted to verify cryopreservation of mouse fibroblasts. The results showed that embryonic cryopreserving method was best one with 0.86 of thawing viability. The embryos from 13-14 d pregnant mouse were superior to 11-12 d and 15-16 d in isolating, growing, laying and living. The first 6 generations were better than following ones in the same aspects above. Cell laying time became longer and vailable time became shorter after the sixth generation. With culture time increasing, fibroblast nuclear size became larger, fibrous filament appeared among fibroblasts, and macrocyst vesicle with fioccule appeared in the cells. Cyst vesicle structure with pyknotic granule appeared in 24 h cultured fibroblasts and macrocyst vesicle also appeared in passaging fibroblasts.

  5. Culture of mouse cardiac microvascular endothelial cells in vitro%小鼠心肌微血管内皮细胞的体外培养

    Institute of Scientific and Technical Information of China (English)

    陈桂秀; 杨明涛; 刘涛; 王浩宇; 刘康; 冯刚

    2013-01-01

    目的 建立小鼠心肌微血管内皮细胞培养体系.方法 4~6周的清洁级C57小鼠的心室肌,利用胰蛋白酶及Ⅱ型胶原酶消化过滤收集的滤液进行重新悬浮种植于明胶包被的培养瓶中,通过倒置电镜观察细胞的生长形态及生长状态,得出生长曲线,并利用免疫荧光鉴定(心肌微血管内皮细胞特异性抗原vWF)培养出的小鼠心肌微血管内皮细胞.结果 通过形态学观察及免疫荧光鉴定证实为小鼠心肌微血管内皮细胞.培养的小鼠心肌微血管内皮细胞第1、2天生长相对缓慢,而到第3、4天细胞呈对数生长,第6、7天细胞达到融合.结论 采用明胶包被培养瓶,通过机械剪切、蛋白酶消化、过滤方法,并进行了相关鉴定,可获得较纯的小鼠心肌微血管内皮细胞,这为研究心肌微血管内皮细胞的迁移、血管再生等提供了实验来源.%Objective To establish the culture system of mouse cardiac microvascular endothelial cells. Methods The ventricular muscle from 4 to 6 weeks of C57 mice was digested by trypsin and type II collagen, then filtered and collected the filtrate to grow in culture bottle that was used gelatin to envelop. The morphology and growth curve of mouse cardiac microvascular endothelial cells was investigated by using electron microscopy. The immunofluorescence was uesd to identify culture of mouse primary cardiac microvascular endothelial cells by the expression of factor vWF-related antigen. Results The mouse cardiac microvascular endothelial cells were demonstrated by morphological observation and immunofluorescence. Culture of mouse cardiac microvascular endothelial cells grow were relatively slowly in 1 to 2 day, grow ogarithmicly in 3 to 4 day, grow cell fusion in 6 to 7 day. Conclusion In the experiment, used gelatin to envelope culture bottle,We can obtain mouse cardiac microvascular endothelial cells with higher purity by mechanical shearing, protease digestion, filtering, and

  6. Effects of Temperature and Water-Filtered Infrared-A Alone or in Combination on Healthy and Glyoxal-Stressed Fibroblast Cultures

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    Lilla Knels

    2012-01-01

    Full Text Available Water-filtered infrared-A (wIRA radiation has been described as supportive for tissue regeneration. We sought to investigate in detail the wIRA effect at different temperatures in 3T3 fibroblasts that were treated with glyoxal to induce formation of advanced glycation end products (AGEs. Nonirradiated and nonglyoxal-treated cells served as controls. Experiments were carried out over a range of 37∘–45∘C with exact temperature monitoring to distinguish between temperature and wIRA effects. Metabolic activity was assessed by resazurin assay. Mitochondrial membrane potential was assessed by JC-1 vital staining. Apoptotic changes were determined by vital staining with annexin V and YO-PRO-1 and determination of subG1 DNA content. Temperature had a dominant effect overriding effects exerted by wIRA or glyoxal treatment. The number of apoptotic cells was significantly higher at 45∘C, while the percentage of healthy cells was significantly lower at 45∘C. WIRA irradiation itself or in combination with glyoxal treatment exerted no damaging effects on the fibroblasts at physiological (37∘–40∘C or higher (42∘–45∘C temperatures compared to untreated controls. Temperatures of 45∘C, which can occur during inappropriate application of infrared irradiation, damage cells even in the absence of wIRA or glyoxal application.

  7. 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.

  8. Velvet antler peptide prevents pressure overload-induced cardiac fibrosis via transforming growth factor (TGF)-β1 pathway inhibition.

    Science.gov (United States)

    Zhao, Lihong; Mi, Yang; Guan, Hongya; Xu, Yan; Mei, Yingwu

    2016-07-15

    Velvet antlers (VAs) are commonly used in traditional Chinese medicine and invigorant and contain many functional components for health promotion. The velvet antler peptide sVAP32 is one of active components in VAs; based on structural study, the sVAP32 interacts with TGF-β1 receptors and disrupts the TGF-β1 pathway. We hypothesized that sVAP32 prevents cardiac fibrosis from pressure overload by blocking TGF-β1 signaling. Sprague-Dawley rats underwent transverse aortic constriction (TAC) or a sham operation. After one month, rats received either sVAP32 (15mg/kg/day) or vehicle for an additional one month. TAC surgery induced significant cardiac dysfunction, fibroblast activation and fibrosis; these effects were improved by treatment with sVAP32. In the heart tissue, TAC remarkably increased the expression of TGF-β1 and connective tissue growth factor (CTGF), reactive oxygen species levels, and the phosphorylation levels of Smad2/3 and extracellular signal-regulated kinases 1/2 (ERK1/2). SVAP32 inhibited the increases in reactive oxygen species levels, CTGF expression and the phosphorylation of Smad2/3 and ERK1/2, but not TGF-β1 expression. In cultured cardiac fibroblasts, angiotensin II (Ang II) had similar effects compared to TAC surgery, such as increases in α-SMA-positive cardiac fibroblasts and collagen synthesis. SVAP32 eliminated these effects by disrupting TGF-β1 binding to its receptors and blocking Ang II/TGF-β1 downstream signaling. These results demonstrated that sVAP32 has anti-fibrotic effects by blocking the TGF-β1 pathway in cardiac fibroblasts.

  9. Isolation and culture of mouse embryonic fibroblasts and preparation of feeder layers%小鼠胚胎成纤维细胞的分离培养及饲养层制备

    Institute of Scientific and Technical Information of China (English)

    胡三强; 王妍妍; 马永宾; 胡嘉波

    2014-01-01

    背景:建立一种既可以大量制备,又易于保存并保持较高活性的饲养层细胞是人胚胎干细胞培养研究的重要环节。  目的:建立昆明小鼠胚胎成纤维细胞的最佳分离培养方法,评价其用于人胚胎干细胞饲养层研究的可行性。方法:用不同浓度胰蛋白酶分步消化法体外分离和培养昆明小鼠胚胎成纤维细胞,观察其生物学特性,制备胚胎成纤维细胞饲养层,检测人胚胎干细胞在饲养层上培养的生长状态。  结果与结论:制备昆明小鼠胚胎成纤维细胞饲养层的最佳胎龄为13.5 d。不同浓度胰蛋白酶分步消化法制备的胚胎成纤维细胞生长状态好,获得的成纤维细胞纯度高,增殖活跃。冻存2周,1,3,6个月内复苏的细胞存活率差异无显著性意义。小鼠胚胎成纤维细胞在第2-4代增殖旺盛,第5代以后细胞增殖活力明显下降。人胚胎干细胞在小鼠胚胎成纤维细胞长期传代后呈典型的未分化形态,碱性磷酸酶和过碘酸-雪夫染色均为阳性。结果表明建立的昆明小鼠胚胎成纤维细胞饲养层分离培养法可为人胚胎干细胞扩增提供稳定、优质的饲养层细胞。%BACKGROUND:It is important to produce and save a large amount of high-activity feeder cel s for the culture of human embryonic stem cel s. OBJECTIVE:To establish the optimal method for isolation and culture of Kunming mouse embryonic fibroblasts, and to evaluate the feasibility of preparing feeder layers for culture of human embryonic stem cel s. METHODS:Embryonic fibroblasts were isolated and cultured by different concentrations of trypsin from Kunming mouse fetuses in vitro. The biological characteristics and growth rule of mouse embryonic fibroblasts were investigated, and then the feeder layers for human embryonic stem cel s culture were produced. The growth of human embryonic stem cel s on the prepared feeder layer was tested

  10. Sitagliptin reduces cardiac apoptosis, hypertrophy and fibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms.

    Directory of Open Access Journals (Sweden)

    Belén Picatoste

    Full Text Available BACKGROUND: Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1 enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart. METHODS: Goto-Kakizaki (GK rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin or vehicle (n=10, each. After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays. RESULTS: Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36, alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells. CONCLUSIONS: Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36 promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actions.

  11. 高糖状态下心肌成纤维细胞Ⅰ、Ⅲ型胶原表达变化的观察%Observation on expression change of typeⅠ and Ⅲ collagen of cardiac fibroblasts under high glucose state

    Institute of Scientific and Technical Information of China (English)

    戴斌; 崔猛; 张宏

    2013-01-01

    目的 探讨高糖对大鼠心肌成纤维细胞Ⅰ、Ⅲ型胶原表达的影响.方法 采用胰酶消化法和差速贴壁分离法获取心肌成纤维细胞,采用正常糖(5.5 mmol/L,对照组)和高糖(25 mmol/L,高糖组)干预.用实时荧光定量PCR(Real-time PCR)方法检测Ⅰ、Ⅲ型前胶原mRNA表达,酶联免疫吸附测定法(ELISA)观察Ⅰ、Ⅲ型胶原蛋白水平的变化.结果 与对照组比较,高糖刺激12 h可使心肌成纤维细胞Ⅰ、Ⅲ型前胶原mRNA表达增加,Ⅰ、Ⅲ前胶原mRNA表达在12 h升高,在24 h达高峰,Ⅰ型前胶原mRNA升高幅度高于Ⅲ型前胶原mRNA;高糖刺激48 h可使Ⅰ、Ⅲ型胶原蛋白表达增加,Ⅰ型胶原蛋白升高幅度高于Ⅲ型胶原.结论 高糖可促进大鼠心肌成纤维细胞合成Ⅰ、Ⅲ型胶原.%Objective To investigate the effect of high glucose on the expression of type Ⅰ and Ⅲ collagen in rat cardiac fibroblasts .Methods Rat cardiac fibroblasts were obtained by pancreatic enzyme digestion method and the differential sidewall separation method .Normal glucose (5 .5 mmol/L, control group) and high glucose(25 mmol/L, high glucose group) were adopted for conducting intervention .The mRNA expressions of type Ⅰ and Ⅲ procollagen were detected by real-time PCR and the changes of type Ⅰ and Ⅲ collagen levels were detected by ELISA .Results Compared with the control group ,the high glucose 12 h stimulation could increase the mRNA expression of Ⅰ and Ⅲ procollagen in rat cardiac fibroblasts .The mRNA expression of collagen was increased at 12 h and reached the peak at 24 h .The increasing amplitude of type Ⅰ collagen mRNA was higher than that of type Ⅲ collagen .The high glucose 48 h stimulation could increase the expression of type Ⅰ and Ⅲ collagen .The increasing amplitude of type Ⅰ collagen was higher than that of collagen type Ⅲ.Conclusion High glucose could promote the synthesis of type Ⅰ and Ⅲ collagen in rat cardiac fibroblasts .

  12. Fibroblast subpopulations in intra-oral wound healing.

    NARCIS (Netherlands)

    Beurden, H.E. van; Snoek, P.A.; Hoff, J.W. Von den; Torensma, R.; Kuijpers-Jagtman, A.M.

    2003-01-01

    The objective of this study was to characterize fibroblasts at sequential time points during intra-oral wound healing in the rat. Experimental wounds were made at several time points in the mucoperiosteum of the palate of 35-day-old Wistar rats. Fibroblasts were cultured from the biopsies under

  13. A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

    Science.gov (United States)

    Igarashi, Junsuke; Okamoto, Ryuji; Yamashita, Tetsuo; Hashimoto, Takeshi; Karita, Sakiko; Nakai, Kozo; Kubota, Yasuo; Takata, Maki; Yamaguchi, Fuminori; Tokuda, Masaaki; Sakakibara, Norikazu; Tsukamoto, Ikuko; Konishi, Ryoji; Hirano, Katsuya

    2016-03-01

    We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

  14. Comparison of protective effect of ascorbic acid on redox and endocannabinoid systems interactions in in vitro cultured human skin fibroblasts exposed to UV radiation and hydrogen peroxide.

    Science.gov (United States)

    Gęgotek, Agnieszka; Bielawska, Katarzyna; Biernacki, Michał; Zaręba, Ilona; Surażyński, Arkadiusz; Skrzydlewska, Elżbieta

    2017-05-01

    The mechanisms of biological activity of commonly used natural compounds are constantly examined. Therefore, the aim of this study was to compare ascorbic acid efficacy in counteracting the consequences of UV and hydrogen peroxide treatment on lipid mediators and their regulative action on antioxidant abilities. Skin fibroblasts exposed to UVA and UVB irradiation, treated with hydrogen peroxide and ascorbic acid. The redox system was estimated through reactive oxygen species (ROS) generation (electron spin resonance spectrometer) and antioxidants level/activity (HPLC/spectrometry) which activity was evaluated by the level of phospholipid metabolites: 4-hydroxynonenal, malondialdehyde, 8-isoprostanes and endocannabinoids (GC/LC-MS) in the human skin fibroblasts. Protein and DNA oxidative modifications were also determined (LC). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), its activators and inhibitors as well as pro/anti-apoptotic proteins and endocannabinoid receptors was examined (Western blot) and collagen metabolism was evaluated by collagen biosynthesis and prolidase activity (spectrometry). UVA and UVB irradiation and hydrogen peroxide treatment enhanced activity of xanthine and NADPH oxidases resulting in ROS generation as well as diminution of antioxidant phospholipid protection (glutathione peroxidase-glutathione-vitamin E), what led to increased lipid peroxidation and decreased endocannabinoids level. Dysregulation of cannabinoid receptors expression and environment of transcription factor Nrf2 caused apoptosis induction. Ascorbic acid partially prevented ROS generation, antioxidant capacity diminution and endocannabinoid systems disturbances but only slightly protected macromolecules such as phospholipid, protein and DNA against oxidative modifications. However, ascorbic acid significantly prevented decrease in collagen type I biosynthesis. Ascorbic acid in similar degree prevents UV (UVA and UVB) and hydrogen peroxide

  15. 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.

  16. Olmesartan ameliorates pressure overload-induced cardiac remodeling through inhibition of TAK1/p38 signaling in mice.

    Science.gov (United States)

    Wu, Lianpin; Mei, Liqin; Chong, Lin; Huang, Yinqing; Li, Yuechun; Chu, Maoping; Yang, Xiangjun

    2016-01-15

    Many studies have demonstrated the potent effects of ARB administration on heart failure. However, the mechanism of the potent effects of ARB on cardiac remodeling is less well understood. We investigated the role of Olmesartan on the fibrosis and hypertrophy in mouse heart. We employed TAC surgery, a mouse model of chronic cardiac failure. All the mice were separated into three groups: the sham group, TAC group and TAC plus Olmesartan group (given Olmesartan treatment after TAC). We analyzed left ventricle remodeling, and function by echocardiography or pathology. We further detected the level of marker genes involved in fibrosis and hypertrophy and in cultured neonatal rat cardiac fibroblasts and myocytes infected by constitutively active TAK1 and p38MAPK. After TAC, all the mice developed hypertrophy, worse cardiac function and malignant remodeling in left ventricle. Olmesartan improved heart remodeling and function without changing pressure of blood. Moreover, Olmesartan reduced the level of transforming growth factor β activated kinase-1 (TAK1) and phospho-p38MAPK. In neonatal rat cardiac fibroblast cells and cardiomyocytes, Olmesartan also decreased TAK1 and p38MAPK activation triggered by TGFβ1 or AngII. The inhibitory effect of Olmesartan was abrogated by overexpression of constitutively active TAK1 and p38MAPK by adenovirus system. Our results suggest Olmesartan improves heart remodeling and function induced by pressure overload. P38MAPK inactivation attenuated by olmesartan via inhibition of TAK1 pathway plays an important role in the process. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Celastrol-Induced Suppression of the MiR-21/ERK Signalling Pathway Attenuates Cardiac Fibrosis and Dysfunction

    Directory of Open Access Journals (Sweden)

    Mian Cheng

    2016-05-01

    Full Text Available Backgroud: Myocardial fibrosis results in myocardial remodelling and dysfunction. Celastrol, a traditional oriental medicine, has been suggested to have cardioprotective effects. However, its underlying mechanism is unknown. This study investigated the ability of celastrol to prevent cardiac fibrosis and dysfunction and explored the underlying mechanisms. Methods: Animal and cell models of cardiac fibrosis were used in this study. Myocardial fibrosis was induced by transverse aortic constriction (TAC in mice. Cardiac hypertrophy and fibrosis were evaluated based on histological and biochemical measurements. Cardiac function was evaluated by echocardiography. The levels of transforming growth factor beta 1 (TGF-β1, extracellular signal regulated kinases 1/2 (ERK1/2 signalling were measured using Western blotting, while the expression of miR-21was analyzed by real-time qRT-PCR in vitro and in vivo. In vitro studies, cultured cardiac fibroblasts (CFs were treated with TGF-β1 and transfected with microRNA-21(miR21. Results: Celastrol treatment reduced the increased collagen deposition and down-regulated α-smooth muscle actin (α-SMA, atrial natriuretic peptide (ANP, brain natriuretic peptides (BNP, beta-myosin heavy chain (β-MHC, miR-21 and p-ERK/ERK. Cardiac dysfunction was significantly attenuated by celastrol treatment in the TAC mice model. Celastrol treatment reduced myocardial fibroblast viability and collagen content and down-regulated α-SMA in cultured CFs in vitro. Celastrol also inhibited the miR-21/ERK signalling pathway. Celastrol attenuated miR-21 up-regulation by TGF-β1 and decreased elevated p-ERK/ERK levels in CFs transfected with miR-21. Conclusion: MiR-21/ERK signalling could be a potential therapeutic pathway for the prevention of myocardial fibrosis. Celastrol ameliorates myocardial fibrosis and cardiac dysfunction, these probably related to miR-21/ERK signaling pathways in vitro and in vivo.

  18. Erythropoietin suppresses the expressions of TGF-β1 and collagen in rat cardiac fibroblasts induced by angiotensin Ⅱ%促红细胞生成素抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞中转化生长因子β1和胶原的表达

    Institute of Scientific and Technical Information of China (English)

    张新金; 马业新; 文渊; 徐雪晶

    2008-01-01

    目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CF)中转化生长因子(TGF)-β1蛋白表达和胶原生成的影响,以及磷脂酰肌醇-3-激酶(PD-K)/Akt信号途径和一氧化氮合酶(NOS)在其中的作用.方法 应用胰酶和胶原酶双酶法分离培养新生大鼠CF细胞,应用EPO、Ang Ⅱ、PI3-K抑制剂LY294002、NOS抑制剂L-NAME等不同因素干预.ELISA法检测CF中Ⅰ型和Ⅲ型胶原的浓度.化学酶法检测CF培养液中的NO浓度以及NOS总的活性及其亚型的活性.Western blot检测Akt、p-Akt、内皮型一氧化氮合酶(eNOS)、iNOS和TGF-β1蛋白的表达.结果 EPO剂量依赖性的抑制Ang Ⅱ诱导的CF培养液中Ⅰ型和Ⅲ型胶原表达以及提高NO的浓度.10 U/ml的EPO对Ⅰ型和Ⅲ型胶原浓度的抑制分别达到了28%和46%,同时NO浓度则提高了154%.EPO也显著抑制了Ang Ⅱ促CF中TGF-β1蛋白的表达,同时Akt的磷酸化水平显著提高,并促进eNOS蛋白的表达.应用LY294002使eNOS蛋白表达水平明显下降,培养液中的NO浓度也随之下降.L-NAME不能降低eNOS蛋白表达,但抑制了NO的生成.EPO抑制Ang Ⅱ诱导的CF中TGF-β1蛋白的表达以及Ⅰ型和Ⅲ型胶原合成作用均能被二者阻断.结论 EPO可抑制Ang Ⅱ诱导的新生大鼠CF中TGF-β1的表达以及Ⅰ型和Ⅲ型胶原表达,可能是通过激活PI3-K/Akt信号途径促使CF中eNOS表达,从而促进NO的表达来实现.%Objective Recent studies have shown cardiac protection effects of erythropoietin (EPO).The present experiment was designed to investigate the effects of EPO on TGF-β1, nitric oxide synthase ( NOS), collagen contents induced by angiotensin Ⅱ ( Ang Ⅱ ) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects.Methods Neonatal rat CFs was isolated by collgenase and trypsinase digestion methods.PBS, EPO, Ang Ⅱ in the absence or presence of LY294002, an inhibitor of PI3-K

  19. Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche.

    Science.gov (United States)

    Pastuła, Agnieszka; Middelhoff, Moritz; Brandtner, Anna; Tobiasch, Moritz; Höhl, Bettina; Nuber, Andreas H; Demir, Ihsan Ekin; Neupert, Steffi; Kollmann, Patrick; Mazzuoli-Weber, Gemma; Quante, Michael

    2016-01-01

    The gastrointestinal epithelium is characterized by a high turnover of cells and intestinal stem cells predominantly reside at the bottom of crypts and their progeny serve to maintain normal intestinal homeostasis. Accumulating evidence demonstrates the pivotal role of a niche surrounding intestinal stem cells in crypts, which consists of cellular and soluble components and creates an environment constantly influencing the fate of stem cells. Here we describe different 3D culture systems to culture gastrointestinal epithelium that should enable us to study the stem cell niche in vitro in the future: organoid culture and multilayered systems such as organotypic cell culture and culture of intestinal tissue fragments ex vivo. These methods mimic the in vivo situation in vitro by creating 3D culture conditions that reflect the physiological situation of intestinal crypts. Modifications of the composition of the culture media as well as coculturing epithelial organoids with previously described cellular components such as myofibroblasts, collagen, and neurons show the impact of the methods applied to investigate niche interactions in vitro. We further present a novel method to isolate labeled nerves from the enteric nervous system using Dclk1-CreGFP mice.

  20. Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation

    Science.gov (United States)

    Ghanbarian, Hossein; Wagner, Nicole; Michiels, Jean-François; Cuzin, François; Wagner, Kay-Dietrich; Rassoulzadegan, Minoo

    2017-01-01

    Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine. PMID:28165496

  1. In vivo fluorescent labeling of corneal wound healing fibroblasts.

    Science.gov (United States)

    Gatlin, Joel; Melkus, Michael W; Padgett, Angela; Petroll, W Matthew; Cavanagh, H Dwight; Garcia, J Victor; Jester, James V

    2003-03-01

    Numerous studies have shown that fibroblasts play an important role in corneal wound healing, however, the dynamic cellular events underlying wound tissue organization and contraction remain unclear. The purpose of this study was to develop a system to enable live cell imaging of corneal wound healing fibroblasts in situ. To this end, concentrated preparations of an RD114 pseudotyped MLV-based vector expressing the enhanced green fluorescent protein (EGFP) were evaluated in vitro for gene transfer efficiency using cultured rabbit corneal keratocytes. Primary rabbit keratocytes were efficiently labeled in vitro (up to 50% EGFP(+)) at a low multiplicity of infection (MOI=10). To evaluate this gene transfer vector in vivo, rabbit corneal fibroblasts were transduced by direct application of vector supernatant to injured corneas following lamellar keratectomy. Fluorescent fibroblasts were then visualized in situ using epifluorescence microscopy and multiphoton confocal microscopy of excised fresh tissue at multiple time points from 14 days to four months following gene transfer. Fourteen days post-transduction, labeled fibroblasts expressing EGFP were readily detectable by fluorescence microscopy. Detectable fluorescence was noted up to eight weeks post-transduction. Labeled fibroblasts were detected in clusters located predominantly along the margin circumscribing the wound and to a lesser extent within the wound area. Cell growth in clusters was suggestive of the expansion of individual transduced clones. High-resolution imaging showed fluorescent fibroblasts to have a broad, flattened, dendritic morphology, distinct from the spi