Project description:Importance of growth factor (GF) signaling in cancer progression is widely acknowledged. Transforming growth factor beta (TGFβ) and and hepa- tocyte growth factor (HGF) are known to play a key role in epithelial-to- mesenchymal transition (EMT) and metastatic cell transformation that are characterized by distinctive alterations in cell mechanical architecture and behavior towards a more robust and motile single cell phenotype. How- ever, molecular mechanisms mediating cancer type specific enhancement of cell mechanical properties in response to growth factors remain unlighted. Here, we combine high-throughput mechanical cell phenotyping, microarray analysis and gene-silencing to dissect molecular mediators of GF-induces cell mechanical enhancement in non-small-cell lung adenocarcinoma (NSCLC). Our experimental results show that both growth factors elevate cell rigidity, size and migratory activity, however, effects of TGFβ stimulation on cell mechanical phenotype and differential gene expression turn out to be significantly stronger. Our microarray analysis reveals that TGFβ-treated NSCLC cells exhibit upregulation of migration-, adhesion-related and down- regulation of cytokinesis-related groups of genes. In addition to vimentin, a canonical marker of EMT, a large number of cytoskeletal and motor proteins including previously reported MYL9, MYLK, TMP1 but also less-known un- conventional myosins are overexpressed in lung adenocarcenoma cells upon treatment with TGFβ. Selective probing of gene-silenced cells lead to identi- fication of unconventional myosin MYH15 as a novel mediator of elevated cell rigidity and invasiveness in TGFβ-stimulated NSCLC cells. In comparison to TGFβ, HGF-treated NSCLC cells respond with upregulation of a relatively small number of genes that are related to actin cyskeleton, microtubules and caveolae. Our results provide insights into phenotypic effects and molecular mechanisms of TGFβ- and HGF-induced mesenchymal cell transformation in NSCLC, and suggest that mediators of cell mechanical enhancement such as unconventional cytoskeletal and motor proteins represent promising phar- maceutical targets for restraining invasive spread of lung cancer.

Project description:Ischemia, fibrosis, and remodeling lead to heart failure after severe myocardial infarction (MI). Myoblast sheet transplantation is a promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in this detrimental disease. We hypothesized that in a rat model of MI-induced chronic heart failure this therapy could further be improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF) in the myoblast sheets. We studied the ability of wild type (L6-WT) and human HGF-expressing (L6-HGF) L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression using microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD) ligation and allowed heart failure to develop for four weeks. Thereafter, we administered L6-WT (n=15) or L6-HGF (n=16) myoblast sheet therapy. Control rats (n=13) underwent LAD ligation and rethoracotomy without therapy and five rats underwent sham-operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy administration. We analyzed cardiac angiogenesis and left ventricular architecture from histological sections 4 weeks after therapy. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further enhanced by hHGF-expression. Analysis of the L6 rat skeletal myoblast cell line and myoblast cell sheets with constitutive human HGF expression.

Project description:Ischemia, fibrosis, and remodeling lead to heart failure after severe myocardial infarction (MI). Myoblast sheet transplantation is a promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in this detrimental disease. We hypothesized that in a rat model of MI-induced chronic heart failure this therapy could further be improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF) in the myoblast sheets. We studied the ability of wild type (L6-WT) and human HGF-expressing (L6-HGF) L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression using microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD) ligation and allowed heart failure to develop for four weeks. Thereafter, we administered L6-WT (n=15) or L6-HGF (n=16) myoblast sheet therapy. Control rats (n=13) underwent LAD ligation and rethoracotomy without therapy and five rats underwent sham-operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy administration. We analyzed cardiac angiogenesis and left ventricular architecture from histological sections 4 weeks after therapy. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further enhanced by hHGF-expression.

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