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James J Tomasek - One of the best experts on this subject based on the ideXlab platform.
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whole animal knockout of smooth muscle alpha actin does not alter excisional wound healing or the fibroblast to Myofibroblast transition
Wound Repair and Regeneration, 2013Co-Authors: James J Tomasek, Robert J Schwartz, Carol J Haaksma, Eric W HowardAbstract:The contractile phenotype and function of Myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized smooth muscle alpha-actin expressed in Myofibroblasts is critical for their formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin -null and wild type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a Myofibroblast-like distribution and a stress fiber-like pattern, demonstrating that these cells acquired the Myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle alpha-actin were expressed in smooth muscle α-actin-null Myofibroblasts, as demonstrated by immunostaining, real-time PCR, and mass spectrometry. These results demonstrate that smooth muscle α-actin is not necessary for Myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in Myofibroblasts.
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myocardin related transcription factors a and b are key regulators of tgf β1 induced fibroblast to Myofibroblast differentiation
Journal of Investigative Dermatology, 2011Co-Authors: Beverly J Crider, Eric W Howard, Carol J Haaksma, George M Risinger, James J TomasekAbstract:Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor β-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into Myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors A and B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates Myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in Myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1-differentiated Myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of Myofibroblast differentiation in response to TGF-β1.
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Contraction of Myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity.
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 2006Co-Authors: James J Tomasek, Melville B. Vaughan, Giulio Gabbiani, Carol J Haaksma, Bradley P. Kropp, Michael D. Martin, Boris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
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contraction of Myofibroblasts in granulation tissue is dependent on rho rho kinase myosin light chain phosphatase activity
Wound Repair and Regeneration, 2006Co-Authors: James J Tomasek, Giulio Gabbiani, Melville Vaugha, Adley P Kropp, Michael D Marti, Carol J Haaksma, Oris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
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Myofibroblasts and mechano-regulation of connective tissue remodelling
Nature Reviews Molecular Cell Biology, 2002Co-Authors: James J Tomasek, Giulio Gabbiani, Boris Hinz, Christine Chaponnier, Robert A. BrownAbstract:During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the Myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the Myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the Myofibroblast — its origins, functions and molecular regulation — will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally. Myofibroblasts are the predominant cell type that are present in granulation tissue of contracting wounds and fibrocontractive diseases, and are also present in some developing or normal adult tissues. The putative function of Myofibroblasts is generating force and altering tissue tension. Myofibroblasts were initially characterized by the presence of microfilament bundles (stress fibres) that are not present in tissue fibroblasts. Two types of Myofibroblasts can be characterized: proto-Myofibroblasts, which contain stress fibres but lack α-smooth muscle (SM) actin, and differentiated Myofibroblasts, which contain both stress fibres and α-SM actin. The formation and maintenance of the proto-Myofibroblast is dependent on isometric tension applied onto a non-compliant substratum. The expression of α-SM actin that is characteristic of the differentiated Myofibroblast is dependent on interaction of ED-A fibronectin with the cell surface and transforming growth factor β1 (TGF-β1). Myofibroblasts in granulation tissue and in in vitro contraction assays generate contractile force in response to certain SM agonists (such as endothelin). Increased expression of α-SM actin is directly correlated with increased force generation by Myofibroblasts. We postulate a positive feedback loop in which tension facilitates TGF-β1 production and/or activation and α-SM actin expression. This, in turn, increases force production and tension development. Myofibroblast contraction is regulated by the level of myosin light chain phosphorylation and the key regulatory step seems to be activation of the Rho–Rho-kinase pathway, which results in the inhibition of myosin light chain phosphatase and increased myosin light chain phosphorylation and contraction. Tissue contraction (contracture) depends on collagen remodelling, a process that is dominated by extracellular-matrix reorganization under the mechanical control of Myofibroblast contraction.
Boris Hinz - One of the best experts on this subject based on the ideXlab platform.
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the Myofibroblast in wound healing and fibrosis answered and unanswered questions
F1000Research, 2016Co-Authors: Marieluce Bochatonpiallat, Giulio Gabbiani, Boris HinzAbstract:The discovery of the Myofibroblast has allowed definition of the cell responsible for wound contraction and for the development of fibrotic changes. This review summarizes the main features of the Myofibroblast and the mechanisms of Myofibroblast generation. Myofibroblasts originate from a variety of cells according to the organ and the type of lesion. The mechanisms of Myofibroblast contraction, which appear clearly different to those of smooth muscle cell contraction, are described. Finally, we summarize the possible strategies in order to reduce Myofibroblast activities and thus influence several pathologies, such as hypertrophic scars and organ fibrosis.
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mechanical aspects of lung fibrosis a spotlight on the Myofibroblast
Proceedings of the American Thoracic Society, 2012Co-Authors: Boris HinzAbstract:Contractile Myofibroblasts are responsible for the irreversible alterations of the lung parenchyma that hallmark pulmonary fibrosis. In response to lung injury, a variety of different precursor cells can become activated to develop Myofibroblast features, most notably formation of stress fibers and expression of α-smooth muscle actin. Starting as an acute and beneficial repair process, Myofibroblast secretion of collagen and contraction frequently becomes excessive and persists. The result is accumulation of stiff scar tissue that obstructs and ultimately destroys lung function. In addition to being a consequence of Myofibroblast activities, the stiffened tissue is also a major promoter of the Myofibroblast. The mechanical properties of scarred lung and fibrotic foci promote Myofibroblast contraction and differentiation. One essential element in this detrimental feed-forward loop is the mechanical activation of the profibrotic growth factor transforming growth factor-β1 from stores in the extracellular ma...
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Myofibroblast contraction activates latent tgf β1 from the extracellular matrix
Journal of Cell Biology, 2007Co-Authors: Pierrejean Wipff, Daniel B Rifkin, Jeanjacques Meister, Boris HinzAbstract:The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile Myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured Myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that Myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of Myofibroblasts and Myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching Myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In Myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated Myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of Myofibroblasts to a stiffened ECM.
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the Myofibroblast one function multiple origins
American Journal of Pathology, 2007Co-Authors: Boris Hinz, Victor J Thannickal, Marieluce Bochatonpiallat, Sem H Phan, Andrea Galli, Giulio GabbianiAbstract:The crucial role played by the Myofibroblast in wound healing and pathological organ remodeling is well established; the general mechanisms of extracellular matrix synthesis and of tension production by this cell have been amply clarified. This review discusses the pattern of Myofibroblast accumulation and fibrosis evolution during lung and liver fibrosis as well as during atheromatous plaque formation. Special attention is paid to the specific features characterizing each of these processes, including the spectrum of different Myofibroblast precursors and the distinct pathways involved in the formation of differentiated Myofibroblasts in each lesion. Thus, whereas in lung fibrosis it seems that most Myofibroblasts derive from resident fibroblasts, hepatic stellate cells are the main contributor for liver fibrosis and media smooth muscle cells are the main contributor for the atheromatous plaque. A better knowledge of the molecular mechanisms conducing to the appearance of differentiated Myofibroblasts in each pathological situation will be useful for the understanding of fibrosis development in different organs and for the planning of strategies aiming at their prevention and therapy.
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formation and function of the Myofibroblast during tissue repair
Journal of Investigative Dermatology, 2007Co-Authors: Boris HinzAbstract:It is generally accepted that fibroblast-to-Myofibroblast differentiation represents a key event during wound healing and tissue repair. The high contractile force generated by Myofibroblasts is beneficial for physiological tissue remodeling but detrimental for tissue function when it becomes excessive such as in hypertrophic scars, in virtually all fibrotic diseases and during stroma reaction to tumors. Specific molecular features as well as factors that control Myofibroblast differentiation are potential targets to counteract its development, function, and survival. Such targets include α-smooth muscle actin and more recently discovered markers of the Myofibroblast cytoskeleton, membrane surface proteins, and the extracellular matrix. Moreover, intervening with Myofibroblast stress perception and transmission offers novel strategies to reduce tissue contracture; stress release leads to the instant loss of contraction and promotes apoptosis.
Giulio Gabbiani - One of the best experts on this subject based on the ideXlab platform.
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the Myofibroblast in wound healing and fibrosis answered and unanswered questions
F1000Research, 2016Co-Authors: Marieluce Bochatonpiallat, Giulio Gabbiani, Boris HinzAbstract:The discovery of the Myofibroblast has allowed definition of the cell responsible for wound contraction and for the development of fibrotic changes. This review summarizes the main features of the Myofibroblast and the mechanisms of Myofibroblast generation. Myofibroblasts originate from a variety of cells according to the organ and the type of lesion. The mechanisms of Myofibroblast contraction, which appear clearly different to those of smooth muscle cell contraction, are described. Finally, we summarize the possible strategies in order to reduce Myofibroblast activities and thus influence several pathologies, such as hypertrophic scars and organ fibrosis.
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the Myofibroblast one function multiple origins
American Journal of Pathology, 2007Co-Authors: Boris Hinz, Victor J Thannickal, Marieluce Bochatonpiallat, Sem H Phan, Andrea Galli, Giulio GabbianiAbstract:The crucial role played by the Myofibroblast in wound healing and pathological organ remodeling is well established; the general mechanisms of extracellular matrix synthesis and of tension production by this cell have been amply clarified. This review discusses the pattern of Myofibroblast accumulation and fibrosis evolution during lung and liver fibrosis as well as during atheromatous plaque formation. Special attention is paid to the specific features characterizing each of these processes, including the spectrum of different Myofibroblast precursors and the distinct pathways involved in the formation of differentiated Myofibroblasts in each lesion. Thus, whereas in lung fibrosis it seems that most Myofibroblasts derive from resident fibroblasts, hepatic stellate cells are the main contributor for liver fibrosis and media smooth muscle cells are the main contributor for the atheromatous plaque. A better knowledge of the molecular mechanisms conducing to the appearance of differentiated Myofibroblasts in each pathological situation will be useful for the understanding of fibrosis development in different organs and for the planning of strategies aiming at their prevention and therapy.
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Contraction of Myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity.
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 2006Co-Authors: James J Tomasek, Melville B. Vaughan, Giulio Gabbiani, Carol J Haaksma, Bradley P. Kropp, Michael D. Martin, Boris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
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contraction of Myofibroblasts in granulation tissue is dependent on rho rho kinase myosin light chain phosphatase activity
Wound Repair and Regeneration, 2006Co-Authors: James J Tomasek, Giulio Gabbiani, Melville Vaugha, Adley P Kropp, Michael D Marti, Carol J Haaksma, Oris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
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the nh2 terminal peptide of α smooth muscle actin inhibits force generation by the Myofibroblast in vitro and in vivo
Journal of Cell Biology, 2002Co-Authors: Boris Hinz, Giulio Gabbiani, Christine ChaponnierAbstract:Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-Myofibroblast modulation is the neo expression of α–smooth muscle actin (α-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in Myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of α-SMA Ac-EEED is delivered to cultured Myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of α–skeletal actin has no effect on Myofibroblasts, whereas the NH2-terminal peptide of β–cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing α-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of α-SMA in wound contraction. The α-SMA–FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.
Carol J Haaksma - One of the best experts on this subject based on the ideXlab platform.
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whole animal knockout of smooth muscle alpha actin does not alter excisional wound healing or the fibroblast to Myofibroblast transition
Wound Repair and Regeneration, 2013Co-Authors: James J Tomasek, Robert J Schwartz, Carol J Haaksma, Eric W HowardAbstract:The contractile phenotype and function of Myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized smooth muscle alpha-actin expressed in Myofibroblasts is critical for their formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin -null and wild type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a Myofibroblast-like distribution and a stress fiber-like pattern, demonstrating that these cells acquired the Myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle alpha-actin were expressed in smooth muscle α-actin-null Myofibroblasts, as demonstrated by immunostaining, real-time PCR, and mass spectrometry. These results demonstrate that smooth muscle α-actin is not necessary for Myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in Myofibroblasts.
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myocardin related transcription factors a and b are key regulators of tgf β1 induced fibroblast to Myofibroblast differentiation
Journal of Investigative Dermatology, 2011Co-Authors: Beverly J Crider, Eric W Howard, Carol J Haaksma, George M Risinger, James J TomasekAbstract:Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor β-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into Myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors A and B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates Myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in Myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1-differentiated Myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of Myofibroblast differentiation in response to TGF-β1.
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Contraction of Myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity.
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 2006Co-Authors: James J Tomasek, Melville B. Vaughan, Giulio Gabbiani, Carol J Haaksma, Bradley P. Kropp, Michael D. Martin, Boris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
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contraction of Myofibroblasts in granulation tissue is dependent on rho rho kinase myosin light chain phosphatase activity
Wound Repair and Regeneration, 2006Co-Authors: James J Tomasek, Giulio Gabbiani, Melville Vaugha, Adley P Kropp, Michael D Marti, Carol J Haaksma, Oris HinzAbstract:During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating Myofibroblasts. We examined whether regulation of Myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured Myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo Myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of Myofibroblasts necessary for wound closure.
Eric W Howard - One of the best experts on this subject based on the ideXlab platform.
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whole animal knockout of smooth muscle alpha actin does not alter excisional wound healing or the fibroblast to Myofibroblast transition
Wound Repair and Regeneration, 2013Co-Authors: James J Tomasek, Robert J Schwartz, Carol J Haaksma, Eric W HowardAbstract:The contractile phenotype and function of Myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized smooth muscle alpha-actin expressed in Myofibroblasts is critical for their formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin -null and wild type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a Myofibroblast-like distribution and a stress fiber-like pattern, demonstrating that these cells acquired the Myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle alpha-actin were expressed in smooth muscle α-actin-null Myofibroblasts, as demonstrated by immunostaining, real-time PCR, and mass spectrometry. These results demonstrate that smooth muscle α-actin is not necessary for Myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in Myofibroblasts.
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myocardin related transcription factors a and b are key regulators of tgf β1 induced fibroblast to Myofibroblast differentiation
Journal of Investigative Dermatology, 2011Co-Authors: Beverly J Crider, Eric W Howard, Carol J Haaksma, George M Risinger, James J TomasekAbstract:Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor β-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into Myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors A and B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates Myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in Myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1-differentiated Myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of Myofibroblast differentiation in response to TGF-β1.
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transforming growth factor β1 promotes the morphological and functional differentiation of the Myofibroblast
Experimental Cell Research, 2000Co-Authors: Melville B. Vaughan, Eric W Howard, James J TomasekAbstract:Abstract The Myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of α-smooth muscle (α-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-β1 (TGF-β1) can promote the expression of α-sm actin in Myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-β1 promoted a dose-dependent increase in the generation of contractile force in Myofibroblasts and a concomitant increase in the expression of α-sm actin. We also demonstrate that TGF-β1 enhanced the formation of the structural elements important in Myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, α-sm actin expression. This differentiated Myofibroblast phenotype was not stable. Removal of TGF-β1 resulted in reduced expression of α-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-β1 promotes the morphological and functional differentiation of the Myofibroblast by first enhancing the formation of the structural elements characteristic of the Myofibroblast followed by increased expression of α-sm actin and contractile force generation.
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regulation of lpa promoted Myofibroblast contraction role of rho myosin light chain kinase and myosin light chain phosphatase
Experimental Cell Research, 2000Co-Authors: Mojgan Parizi, Eric W Howard, James J TomasekAbstract:Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted Myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating Myofibroblast contraction. In addition, the regulation of Myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted Myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote Myofibroblast contraction. These results suggest that Ca2+-mediated activation of MLCK, while necessary, is not sufficient to promote Myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted Myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote Myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted Myofibroblast contraction, in contrast to smooth muscle cells in which Ca2+ activation of MLCK alone is sufficient to promote contraction.
