Zhang, Yang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 179-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts. / May 2008

Thesis (M.S.), Biology--University of Central Oklahoma, 2009. / Includes bibliographical references (leaves 53-57).

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts.

Fibroblast growth factor-2 (FGF-2) is expressed as high molecular weight (> 20 kDa, Hi-FGF-2), or low molecular weight, (18 kDa, Lo-FGF-2) isoforms with distinct functions in the heart and other tissues. Studies to-date have focused on Lo-FGF-2, while the biology of Hi-FGF-2 is less well understood. This work investigated potential autocrine and paracrine effects of rat and human Hi-FGF-2 on cardiac myocytes and non-myocytes (myofibroblasts). Using rat ventricular myofibroblast cultures stimulated with angiotensin II (Ang II), in the absence or presence of YVAD, a peptide inhibitor of caspase-1, it was shown that caspase-1 activity was required for the Ang II-stimulated Hi-FGF-2 secretion. Secreted rat Hi-FGF-2 was shown to be biologically active and capable of stimulating neonatal as well as adult cardiomyocyte hypertrophy in vitro. The effect of extracellular-acting Hi- versus Lo-FGF-2 on the secretome profile of rat cardiac myofibroblasts was compared. Conditioned media collected after stimulation with rat Hi- or Lo-FGF-2 were analyzed by mass spectroscopy (LC-MS/MS). Secretome profiles suggested that Hi-FGF-2 was more potent than Lo-FGF-2 in upregulating several matricellular and fibrosis-associated proteins, most prominently periostin, follistatin-like protein 1, plasminogen activator inhibitor-1, and tenascin. Human heart (atrial) tissue, pericardial fluid, and human heart-derived myofibroblasts were shown to accumulate predominantly Hi-FGF-2. Ang II up-regulated Hi-FGF-2 in human cells, via activation of: type 1 or type 2 Ang II receptors (AT-1R, AT-2R); the ERK pathway; and matrix metalloprotease-2. Neutralizing antibodies specific for Hi-FGF-2 (neu-AbHi-FGF-2) reduced expression of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis. Blocking the autocrine action of Hi-FGF-2 on human cells with neu-AbHi-FGF-2 resulted in down-regulation of periostin, as well as α-smooth muscle actin, pro-collagen, embryonic smooth muscle myosin, and extra domain A fibronectin, consistent with a reversal from activated myofibroblast to fibroblast phenotype. Stimulation of human myofibroblasts with human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating pro-interleukin-1β and plasminogen activator inhibitor-1, considered to be pro-inflammatory proteins. It is concluded that exported, extracellular-acting Hi-FGF-2 has pro-fibrotic, pro-inflammatory, and pro-hypertrophic properties, contributes to the ‘activated fibroblast’ phenotype, and represents a therapeutic target for prevention of maladaptive cardiac remodeling in humans.

The incidence of cardiovascular disease has reached epidemic proportions in North America. Specifically, myocardial infarctions (MI) are a major contributor to heart failure which greatly influences morbidity and mortality rates in developed nations. In the post-MI heart, cardiac fibroblasts migrate to the damaged area, convert to myofibroblasts and contribute to infarct scar contraction. As well, cardiac myofibroblasts are hypersynthetic for matrix components eg, collagen, and de novo production of fibrillar collagens lessens the chance for acute scar rupture. TGF-1 is important in the initiation of cardiac healing and fibrosis. Canonical TGF-1 signaling occurs with the activation of receptor-operated Smads (R-Smads) including Smad3. The current study addresses the question of whether Smad3 and/or myocardin influence myofibroblast contractility. We believe that myocardin is a Smad3 binding partner and cofactor and thus contributes to Smad associated healing and fibrotic events in the heart. In mesenchyme-derived cells, myocardin exists as a nuclear protein and is a cardiac and smooth muscle specific transcriptional coactivator of serum response factor (SRF). This transcription factor has been shown to bind to Smad3 in COS-7 cells (a green monkey kidney fibroblast-like cell line) and we suggest that it may contribute to fibroproliferative events. Precisely how Smad3/myocardin facilitates post-MI wound healing and/or contributes to inappropriate post-MI fibrosis is unknown. Very little work has been done to address myocardin expression in cardiac ventricular myofibroblasts. While a number of previous studies address TGF-β/Smad signaling in cardiac myofibroblasts, none have addressed the effects of overexpressed Smad3 on cellular contractility and collagen secretion. As Smad3 and its endogenous inhibitor Smad eg, I-Smad7, contribute significantly to TGF-β signaling in myofibroblasts, we rationalize that they must be important in the regulation of many fibroproliferative processes. Our goals were first to measure/determine myocardin expression in primary ventricular myofibroblasts; second, to explore a putative interaction between Smad3 and myocardin; third to examine a possible link between TGF-β1 stimulation, myocardin and Smad3. Finally, we sought to examine the effect of overexpressed Smad3, Smad7 and myocardin on contractility and collagen production. These experiments were conducted by using RT-PCR, co-immunoprecipitation, adenoviral overexpression of Smad3, Smad7 and myocardin, Western blot analysis, collagen gel deformation assays (contractility studies) and finally, Pro-collagen 1 N-terminal Peptide (P1NP) secretion as a measure of mature collagen production. We document the novel expression of myocardin in ventricular myofibroblasts and provide evidence that myocardin may serve as a Smad3 cofactor in cardiac myofibroblasts. Further, myocardin overexpression is linked to increased contractility in myofibroblasts compared to LacZ infected controls, and that TGF-β1 acutely stimulated myocardin expression followed by a dramatic reduction 1 hour thereafter. Overexpressed Smad3 alone led to increased contractility in primary ventricular myofibroblasts. Thus the effect of increasing myocardin expression had a comparable effect to that of increased Smad3 alone with this endpoint. Finally, overexpression of both Smad3 and myocardin in the presence of TGF-β1 led to an additive stimulation of contractility in cells when compared to the effect of TGF-β1 stimulation alone. Overexpressed Smad7 alone was associated with decreased secretion of type I collagen when compared to the control; when cells overexpressing Smad7 are stimulated with TGF-β1, collagen secretion is dramatically reduced when compared to cells treated with TGF-β1. In an addition series of experiments we addressed reverse mode NCX1 function as a means of Ca2+ entry to the cytosol of myofibroblasts upon their excitation. We have previously shown the stimulatory effect of TGF-β1 on myofibroblast contractility, and we now report that overexpression of Smad3 alone led to increased mRNA expression of NCX1. Thus it is possible that TGF-β1 signaling via Smad3 may influence Ca2+ movements and thus contractile performance in ventricular myofibroblasts.

Thesis (M.Sc. Materials and Process Engineering)--University of Waikato, 2006. / Title from PDF cover (viewed April 23, 2009) Includes bibliographical references (p. 100-104)

The display of Lithuanian mare’s endometritis and endometrosis was investigated. The prevalence of myofibroblasts and tenascin was determined during endometritis and endometrosis. An acute phase protein serum amyloid A studies in mares’ endometrium were carried out. The changes of the vessels walls thickness and the amount of elastic fibers, due to mares’ age and endometrial pathology, were evaluated using morfometric analysis.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.