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Philippe Menasche - One of the best experts on this subject based on the ideXlab platform.
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cell delivery intramyocardial injections or epicardial deposition a head to head comparison
The Annals of Thoracic Surgery, 2009Co-Authors: Hadhami Hamdi, Valérie Bellamy, Séverine Peyrard, Onnik Agbulut, Akira Furuta, Alain Bel, Etienne Puymirat, Philippe MenascheAbstract:Background Multiple needle-based injections of cells in the myocardium are associated with a low engraftment rate, which may limit the benefits of the procedure. This study used Skeletal Myoblasts to perform a head-to-head comparison of conventional injections with epicardial deposition of scaffold-embedded cells. Methods Four weeks after ligation-induced myocardial infarction, 40 rats were randomly allocated to receive intramyocardial injections of 5 million human Skeletal Myoblasts or control medium or to have the infarcted area covered with either a bilayer myoblast cell sheet prepared from a fibrin-coated culture plate or a myoblast-seeded collagen sponge (Gelfoam; Pharmacia & Upjohn, Kalamazoo, MI). End points, assessed after 1 month, included left ventricular function blindly measured by echocardiography, quantification of cell engraftment by quantitative real-time polymerase chain reaction and immunostaining, histologic assessment of fibrosis and angiogenesis, and tissue levels of host-specific angiogenic and antifibrotic cytokines. Results Compared with control medium- or myoblast-injected hearts, those receiving the two cell constructs demonstrated the highest recoveries of left ventricular function ( p = 0.004 versus controls). Both myoblast cell sheets and myoblast-seeded Gelfoam sponges also resulted in significantly greater angiogenesis compared with controls. The Gelfoam group was associated with the best outcome with regard to the number of engrafted donor cells ( p = 0.03 versus Myoblasts) and the reduction of fibrosis ( p = 0.02 and p = 0.04 versus the control and myoblast groups, respectively). Conclusions Compared with injections, delivery of Myoblasts in a construct overlaying the infarcted area is associated with better graft functionality, possibly because of maintenance of improved cell patterning. The cell-seeded Gelfoam construct was found to feature a user-friendly, reproducible, and atraumatic technique.
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characterization of the paracrine effects of human Skeletal Myoblasts transplanted in infarcted myocardium
European Journal of Heart Failure, 2008Co-Authors: Maitane Perezilzarbe, A Hagege, Michel Desnos, Onnik Agbulut, Beatriz Pelacho, Cristina Ciorba, Edurne San Joseeneriz, Pablo Aranda, Enrique J Andreu, Philippe MenascheAbstract:Background: The discrepancy between the functional improvements yielded experimentally by Skeletal Myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms. Methods and results: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56 + ), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56 � ). Gene expression revealed up-regulation of pro-angiogenic (PGF), antiapoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (pb0.05) and a decrease in apoptosis of cardiomyocytes (pb0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-β, and IGF-1 1 month after transplant. Conclusions: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.
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Skeletal Myoblasts and cardiac repair
Journal of Molecular and Cellular Cardiology, 2008Co-Authors: Philippe MenascheAbstract:The seminal experiments showing that cells transplanted in infarcted hearts could effect myocardial tissue repair have provided the proof of concept that cell therapy might be an effective means of improving the outcome of patients with severe heart failure. Because of their appealing characteristics (autologous origin, in vitro scalability, high resistance to ischemia), Skeletal Myoblasts have undergone extensive preclinical testing that has consistently demonstrated their ability to preserve postinfarct left ventricular function and to limit remodelling. As this functional efficacy occurs despite a poor long-term engraftment rate and the inability of Myoblasts to convert into cardiomyocytes, the hypothesis has been raised that the predominant mechanism of action could involve paracrine signalling rather than a direct contractile effect of the graft. These preclinical data have paved the way for the early human trials which have confirmed the feasibility and safety of this approach. The mixed results in terms of efficacy should not be discouraging; they only reflect that the field is still in infancy and have yet been helpful in identifying some key issues like the limited efficiency of current cell transfer techniques and the high rate of early posttransplantation cell death. It is clear, however, that Myoblasts and, more generally, adult stem cells cannot truly repair infarcted myocardium through the generation of new cardiomyocytes. This first wave of clinical studies thus delineates the research pathways that need to be followed for overcoming these hurdles and consequently allow myoblast transplantation to become a potentially effective adjunct to current heart failure therapies.
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Skeletal Myoblasts preserve remote matrix architecture and global function when implanted early or late after coronary ligation into infarcted or remote myocardium
Circulation, 2008Co-Authors: Patrick Farahmand, Teresa Y Y Lai, Richard D Weisel, Shafie Fazel, Terrence M Yau, Philippe MenascheAbstract:Background— The inability of Skeletal Myoblasts to transdifferentiate into cardiomyocytes suggests that their beneficial effects on cardiac function after a myocardial infarction are mediated by paracrine effects. We evaluated the roles of these factors in the preservation of matrix architecture (in the infarct and remote regions) by varying the timing (postmyocardial infarction) and delivery site of the implanted cells. Methods and Results— Skeletal Myoblasts (5×106) or control media were injected into the infarct or noninfarcted myocardium at 5 or 30 days after coronary artery ligation in rats. Function was assessed by echocardiography before transplantation and 14 and 30 days thereafter and with a Millar catheter at 30 days after transplantation. Ventricular geometry, remote fibrillar collagen architecture, and changes in the matrix metalloproteinase-TIMP system were evaluated. Myoblast implantation in both sites and at both times preserved matrix architecture (length and width of collagen fibers) in t...
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Self-assembling peptide nanofibers and Skeletal myoblast transplantation in infarcted myocardium.
Journal of biomedical materials research. Part B Applied biomaterials, 2008Co-Authors: Gilbert Dubois, A Hagege, Patrick Bruneval, Valérie Bellamy, Séverine Peyrard, Laurent Sabbah, Vincent F. M. Segers, Richard T. Lee, Philippe MenascheAbstract:Cell transplantation is currently limited by poor graft retention and survival in the postinfarction scar. Because this issue could potentially be addressed by embedding cells in bioinjectable scaffolds and boosting cell survival pathways, we induced a myocardial infarction in 72 rats to assess the effects of different self-assembling peptides with or without platelet-derived growth factor (PDGF-BB) on survival of transplanted Skeletal Myoblasts. Two weeks after coronary artery ligation, rats were randomized to receive in-scar injections of culture medium (controls, n = 11), self-assembling peptide (RAD16-I) nanofibers (NF, n = 9), Skeletal Myoblasts (n = 12), or Skeletal Myoblasts in combination with NF (n = 8). In separate experiments with different self-assembling peptides (RAD16-II), rats received in-scar injections of culture medium (controls, n = 6), Skeletal Myoblasts (n = 10), PDGF-loaded peptides (n = 7), or Skeletal Myoblasts (5 x 10(6)) in combination with PDGF-loaded peptides (n = 9). After 1 month, left ventricular function, as assessed by echocardiography, was not improved in either of the experimental groups compared with controls. This correlated with the failure of RAD16-I peptides or PDGF-loaded RAD16-II peptides to improve myoblast survival despite a greater angiogenesis. In vitro experiments confirmed that the number of Myoblasts decreased over time when seeded on nanofiber gels. These data suggest that the optimal use of biomaterial scaffolds for survival of transplanted cells will require specific tailoring of the biomaterial to the cell type.
Doris A Taylor - One of the best experts on this subject based on the ideXlab platform.
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engineering Skeletal Myoblasts roles of three dimensional culture and electrical stimulation
American Journal of Physiology-heart and Circulatory Physiology, 2005Co-Authors: Dawn Pedrotty, Doris A Taylor, Bryce H Davis, Patrick D Wolf, Laura E NiklasonAbstract:Immature Skeletal muscle cells, or Myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studi...
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engineering Skeletal Myoblasts roles of three dimensional culture and electrical stimulation
American Journal of Physiology-heart and Circulatory Physiology, 2005Co-Authors: Dawn Pedrotty, Doris A Taylor, Jennifer Koh, Bryce H Davis, Patrick D Wolf, Laura E NiklasonAbstract:Immature Skeletal muscle cells, or Myoblasts, have been used in cellular cardiomyoplasty in attempts to regenerate cardiac muscle tissue by injection of cells into damaged myocardium. In some studies, muscle tissue within myoblast implant sites may be morphologically similar to cardiac muscle. We hypothesized that identifiable aspects of the cardiac milieu may contribute to growth and development of implanted Myoblasts in vivo. To test this hypothesis, we designed a novel in vitro system to mimic some aspects of the electrical and biochemical environment of native myocardium. This system enabled us to separate the three-dimensional (3-D) electrical and biochemical signals that may be involved in myoblast proliferation and plasticity. Myoblasts were grown on 3-D polyglycolic acid mesh scaffolds under control conditions, in the presence of cardiac-like electrical current fluxes, or in the presence of culture medium that had been conditioned by mature cardiomyocytes. Cardiac-like electrical current fluxes caused increased myoblast number in 3-D culture, as determined by DNA assay. The increase in cell number was due to increased cellular proliferation and not differences in apoptosis, as determined by proliferating cell nuclear antigen and TdT-mediated dUTP nick-end labeling. Cardiomyocyte-conditioned medium also significantly increased myoblast proliferation. Expression of transcription factors governing differentiation along Skeletal or cardiac lineages was evaluated by immunoblotting. Although these assays are qualitative, no changes in differentiation state along Skeletal or cardiac lineages were observed in response to electrical current fluxes. Furthermore, from these experiments, conditioned medium did not appear to alter the differentiation state of Skeletal Myoblasts. Hence, cardiac milieu appears to stimulate proliferation but does not affect differentiation of Skeletal Myoblasts.
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comparison of intracardiac cell transplantation autologous Skeletal Myoblasts versus bone marrow cells
Circulation, 2003Co-Authors: Richard B Thompson, Donald D Glower, Bryce H Davis, Sitaram M Emani, Ewout J Van Den Bos, Yoshihisa Morimoto, Damian M Craig, Doris A TaylorAbstract:Anincreasing number of patients living with cardiovascular disease (CVD) and still unacceptably high mortality created an urgent need to effectively treat and prevent disease-related events. Within the past 5 years, Skeletal Myoblasts (SKMBs) and bone marrow (or blood)-derived mononuclear cells (BMNCs) have demonstrated preclinical efficacy in reducing ischemia and salvaging already injured myocardium, and in preventing left ventricular (LV) remodeling, respectively. These findings have been translated into clinical trials, so far totaling over 200 patients for SKMBs and over 800 patients for BMNCs. These safety/feasibility and early phase II studies showed promising but somewhat conflicting symptomatic and functional improvements, and some safety concerns have arisen. However, the patient population, cell type, dose, time and mode of delivery, and outcome measures differed, making comparisons problematic. In addition, the mechanisms through which cells engraft and deliver their beneficial effects remain to be fully elucidated. It is now time to critically evaluate progress made and challenges encountered in order to select not only the most suitable cells for cardiac repair but also to define appropriate patient populations and outcome measures. Reiterations between bench and bedside will increase the likelihood of cell therapy success, reduce the time to development of combined of drug- and cell-based disease management algorithms, and offer these therapies to patients to achieve a greater reduction of symptoms and allow for a sustained improvement of quality of life.
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endoventricular transplantation of allogenic Skeletal Myoblasts in a porcine model of myocardial infarction
Journal of Endovascular Therapy, 2002Co-Authors: Nabil Dib, Edward B Diethrich, Ann Campbell, Noreen Goodwin, Barb Robinson, James Gilbert, Dan W Hobohm, Doris A TaylorAbstract:Purpose:To assess the technical feasibility of percutaneous endoventricular injection of Skeletal Myoblasts into an infarcted porcine myocardium.Methods:A Skeletal muscle biopsy was obtained from a donor pig and processed for myoblast expansion in vitro. Myocardial infarction was induced in a host pig via fibrin coil placement in the left anterior descending artery. Four weeks later, electromechanical mapping of the left ventricle identified the infarction site, into which ∼200 million allogenic cells obtained from the muscle biopsy were directly injected (0.1 mL/injection at 25 sites) from inside the ventricular cavity via a needle injection catheter inserted through the femoral artery. Ten days after transplantation, the injected heart was evaluated histologically for the presence of Myoblasts.Results:Electrocardiography, echocardiography, left ventricular angiography, and electromechanical mapping confirmed the myocardial infarction. During the cell transfer procedure, premature ventricular contraction...
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Cellular cardiomyoplasty with autologous Skeletal Myoblasts for ischemic heart disease and heart failure
Trials, 2001Co-Authors: Doris A TaylorAbstract:Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Even though it is based on many years of pre-clinical studies, much remains to be understood about this methodology, even as it progresses to the clinic. For example, controversies exist over the specific cells to be used, the dosages needed for tissue repair, how cells will affect the electrical activity of the myocardium, and even whether the cells can improve myocardial function after transplantation — all of which are briefly reviewed here. Autologous Skeletal Myoblasts appear to be the most well studied and best first generation cells for cardiac repair. Yet cardiocytes and, more recently, stem cells have been proposed as cell sources for this technology. Their advantages and limitations are also discussed. Although cellular cardiomyoplasty (cell transplantation for cardiac repair) shows great pre-clinical promise, its future will heavily depend on conducting carefully controlled, randomized clinical trials with appropriate endpoints. Utilizing biologically active cells provides both an opportunity for tissue repair and the potential for not yet understood outcomes. As with any frontier, many pioneers will attempt to conquer it. But also as with any frontier, there are pitfalls and consequences to be considered that may surpass those of previous endeavors. The future thus requires careful consideration and well-designed trials rather than haste. The promise for cell transplantation is too great to be spoiled by ill-designed attempts that forget to account for the biology of both the cells and the myocardium.
Claire E Stewart - One of the best experts on this subject based on the ideXlab platform.
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tumor necrosis factor induced apoptosis is associated with suppression of insulin like growth factor binding protein 5 secretion in differentiating murine Skeletal Myoblasts
Journal of Cellular Physiology, 2000Co-Authors: Kate A Meadows, Jeffrey M P Holly, Claire E StewartAbstract:Wasting of muscle and fat during cachexia exceeds that explained by reduced food intake alone. This wasting may result from an imbalanced cytokine environment, which could lead to increased protein catabolism. Supporting this, tumor necrosis factor-α (TNF-α) is raised in several animal models of cachectic muscle wasting. Therefore, we assessed the effects of TNF-α and its second messenger, ceramide, on the proliferation, differentiation, and survival of murine C2 Skeletal Myoblasts. Because insulin-like growth factor binding protein-5 (IGFBP-5) and insulin-like growth factor-II (IGF-II) are potent regulators of myoblast proliferation and differentiation, we monitored the ability of exogenous TNF-α to manipulate this system. Fibroblast growth factor (FGF) ceramide, or TNF-α suppressed differentiation of C2 cells compared with controls. All treatments suppressed IGF-II production but only TNF-α blocked IGFBP-5 secretion. TNF-α increased apoptotic cell death, which otherwise remained basal (low serum differentiation medium (LSM), FGF) or low (ceramide). Suppression of both IGFBP-5 and IGF-II secretion may explain why of all triggers tested, only TNF-α not only blocked differentiation, but also promoted cell death. This suggests a fundamental role of IGFBP-5 for maintaining muscle survival. Supporting this hypothesis, no increase in apoptosis was seen in IGFBP-5 cDNA tranfected C2 cells after TNF-α treatment. In summary, the IGF system is essential for maintaining Skeletal muscle cell survival and differentiation, and its suppression by TNF-α is fundamental regarding muscle wasting, and may be associated in vivo with cancer cachexia. J. Cell. Physiol. 183:330–337, 2000. © 2000 Wiley-Liss, Inc.
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tumor necrosis factor alpha induced apoptosis is associated with suppression of insulin like growth factor binding protein 5 secretion in differentiating murine Skeletal Myoblasts
Journal of Cellular Physiology, 2000Co-Authors: Kate A Meadows, Jeffrey M P Holly, Claire E StewartAbstract:Wasting of muscle and fat during cachexia exceeds that explained by reduced food intake alone. This wasting may result from an imbalanced cytokine environment, which could lead to increased protein catabolism. Supporting this, tumor necrosis factor-α (TNF-α) is raised in several animal models of cachectic muscle wasting. Therefore, we assessed the effects of TNF-α and its second messenger, ceramide, on the proliferation, differentiation, and survival of murine C2 Skeletal Myoblasts. Because insulin-like growth factor binding protein-5 (IGFBP-5) and insulin-like growth factor-II (IGF-II) are potent regulators of myoblast proliferation and differentiation, we monitored the ability of exogenous TNF-α to manipulate this system. Fibroblast growth factor (FGF) ceramide, or TNF-α suppressed differentiation of C2 cells compared with controls. All treatments suppressed IGF-II production but only TNF-α blocked IGFBP-5 secretion. TNF-α increased apoptotic cell death, which otherwise remained basal (low serum differentiation medium (LSM), FGF) or low (ceramide). Suppression of both IGFBP-5 and IGF-II secretion may explain why of all triggers tested, only TNF-α not only blocked differentiation, but also promoted cell death. This suggests a fundamental role of IGFBP-5 for maintaining muscle survival. Supporting this hypothesis, no increase in apoptosis was seen in IGFBP-5 cDNA tranfected C2 cells after TNF-α treatment. In summary, the IGF system is essential for maintaining Skeletal muscle cell survival and differentiation, and its suppression by TNF-α is fundamental regarding muscle wasting, and may be associated in vivo with cancer cachexia. J. Cell. Physiol. 183:330–337, 2000. © 2000 Wiley-Liss, Inc.
Michael P. Lisanti - One of the best experts on this subject based on the ideXlab platform.
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expression of caveolin 3 in Skeletal cardiac and smooth muscle cells caveolin 3 is a component of the sarcolemma and co fractionates with dystrophin and dystrophin associated glycoproteins
Journal of Biological Chemistry, 1996Co-Authors: Kenneth S Song, Philipp E. Scherer, Zhaolan Tang, Takashi Okamoto, Shengwen Li, Mark Chafel, Stave D Kohtz, Michael P. LisantiAbstract:Abstract Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21–24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within Skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and Skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in Skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 Skeletal Myoblasts in culture. Using differentiated C2C12 Skeletal Myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, α-sarcoglycan, and β-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.
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expression of caveolin 3 in Skeletal cardiac and smooth muscle cells caveolin 3 is a component of the sarcolemma and co fractionates with dystrophin and dystrophin associated glycoproteins
Journal of Biological Chemistry, 1996Co-Authors: Kenneth S Song, Philipp E. Scherer, Zhaolan Tang, Takashi Okamoto, Mark Chafel, Stave D Kohtz, Caryn Chu, Michael P. LisantiAbstract:Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within Skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and Skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in Skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 Skeletal Myoblasts in culture. Using differentiated C2C12 Skeletal Myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, alpha-sarcoglycan, and beta-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.
Stave D Kohtz - One of the best experts on this subject based on the ideXlab platform.
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expression of caveolin 3 in Skeletal cardiac and smooth muscle cells caveolin 3 is a component of the sarcolemma and co fractionates with dystrophin and dystrophin associated glycoproteins
Journal of Biological Chemistry, 1996Co-Authors: Kenneth S Song, Philipp E. Scherer, Zhaolan Tang, Takashi Okamoto, Shengwen Li, Mark Chafel, Stave D Kohtz, Michael P. LisantiAbstract:Abstract Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21–24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within Skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and Skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in Skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 Skeletal Myoblasts in culture. Using differentiated C2C12 Skeletal Myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, α-sarcoglycan, and β-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.
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expression of caveolin 3 in Skeletal cardiac and smooth muscle cells caveolin 3 is a component of the sarcolemma and co fractionates with dystrophin and dystrophin associated glycoproteins
Journal of Biological Chemistry, 1996Co-Authors: Kenneth S Song, Philipp E. Scherer, Zhaolan Tang, Takashi Okamoto, Mark Chafel, Stave D Kohtz, Caryn Chu, Michael P. LisantiAbstract:Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within Skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and Skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in Skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 Skeletal Myoblasts in culture. Using differentiated C2C12 Skeletal Myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, alpha-sarcoglycan, and beta-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.
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positive and negative regulation of d type cyclin expression in Skeletal Myoblasts by basic fibroblast growth factor and transforming growth factor β a role for cyclin d1 in control of myoblast differentiation
Journal of Biological Chemistry, 1995Co-Authors: Sunkara S Rao, Stave D KohtzAbstract:Abstract Differentiation of Skeletal Myoblasts in culture is negatively regulated by certain growth factors, including basic fibroblast growth factor (bFGF) and transforming growth factor β (TGFβ). We investigated the effects of bFGF and TGFβ on D-type cyclin expression in Skeletal Myoblasts. When Myoblasts were induced to differentiate in low mitogen medium, expression of cyclin D1 rapidly fell below detectable levels. In contrast, expression of cyclin D3 increased to levels exceeding those present in Myoblasts. Expression of cyclin D1 was induced in Myoblasts by bFGF and TGFβ (albeit with different kinetics for each factor), while induction of cyclin D3 expression was inhibited by these growth factors. Although these results are consistent with other reports showing induction of cyclin D1 by growth factors, induction of cyclin D3 expression during terminal differentiation of Myoblasts and inhibition of this induction by growth factors is surprising. These results suggest that cyclin D3, previously thought to be only a positive regulator of cell cycle progression, may also function in the cellular context of terminal differentiated muscle. Stable expression of cyclin D1 from an ectopic viral promoter inhibits C2C12 myoblast differentiation, but only in those clones where the level of cyclin D1 expression does not significantly exceed that present in control Myoblasts stimulated by bFGF. Together, these result suggest that cyclin D1 expression functions in the inhibition of myoblast differentiation by certain growth factors.
