No description available.

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.

No description available.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses. / May 2006

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme expressed in highly proliferating cells. Studies of PKM2 have been focused on its function of promoting cell proliferation in cancer cells. Our laboratory previously discovered that extracellular PKM2 released from cancer cells promoted angiogenesis by activating endothelial cell proliferation and migration. PKM2 activated endothelial cells through integrin αvβ3. Angiogenesis and myofibroblast differentiation are key processes during wound healing. In this dissertation, I demonstrate that extracellular PKM2 released from activated neutrophils promotes angiogenesis and myofibroblast differentiation during wound healing. PKM2 activates dermal fibroblasts through integrin αvβ3 and PI3K signaling pathway. I also claim that extracellular PKM2 plays a role during liver fibrosis. PKM2 protects hepatic stellate cells from apoptosis by activating the survival signaling pathway.

Cardiac fibrosis contributes to heart failure by dramatically impairing cardiac function, increasing patient morbidity and mortality. The primary fibrillar collagen expressed in the heart is type I, and increased collagen synthesis is the hallmark of cardiac fibrosis. Our laboratory has shown that the transcription factor scleraxis is sufficient to regulate the gene encoding collagen Iα2. The present thesis identifies and focuses on three key functions of scleraxis in the heart. First, we show that scleraxis is required for production of the cardiac extracellular matrix. Using in vitro and in vivo models, we observed a significant upregulation/reduction of matrix genes in response to induction/loss of scleraxis gene function respectively. In fact, scleraxis overexpression was sufficient to rescue matrix synthesis in scleraxis-null cells. In a murine model of cardiac pressure overload, scleraxis gene deletion blunted the induction of fibrotic collagen gene expression. Second, we provide evidence that scleraxis governs fibroblast-myofibroblast phenotype transition and fibroblast number. Scleraxis gene induction promoted cardiac myofibroblast phenoconversion while knockdown reduced myofibroblast marker gene expression. Scleraxis exerts direct transcriptional control on the a-smooth muscle actin gene-an established marker of myofibroblasts. Scleraxis null mice exhibited a dramatic reduction in cardiac fibroblast numbers- this is attributed to impairment of the epithelial-to-mesenchymal transition program which was marked by a corresponding loss of mesenchymal markers and increased epithelial markers. Loss-of-function experiments using primary cardiac proto-myofibroblasts recapitulated this paradigm, whereas scleraxis gene induction showed a reciprocal effect on mesenchymal markers. Third, data from this study supports the required role of scleraxis in the TGFb/Smad signaling pathway. Scleraxis is strongly upregulated by the potent pro-fibrotic cytokine TGFb, and works synergistically with the canonical Smad signaling pathway to increase Col1a2 expression by cardiac fibroblasts and myofibroblasts. Smad3 induced expression of the fibrillar collagens – an effect that was significantly attenuated following scleraxis knockdown. Smad3 binding to the Col1a2 gene promoter was significantly reduced in scleraxis null hearts. This study involved a comprehensive series of in vitro and in vivo experiments, and is the first to identify scleraxis as a key regulator of multiple fibroblast functions and a potential future target for therapeutic intervention in cardiac fibrosis. / May 2016

No description available.