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Molecular mechanisms involved in TGF-?1-induced Muscle-derived stem cells differentiation to smooth muscle cells.

ABSTRACT: We investigated the molecular mechanisms involved in transforming growth factor beta 1 (TGF-?1)-induced myogenic stem cell differentiation to smooth muscle cells. We isolated muscle-derived stem cells (MDSCs) from gastrocnemius muscles following their identification by immunohistochemistry analysis of desmin and flow cytometry analysis of SCA-1, CD34, and CD45. MDSCs at passage 3 (PP3) were cultured in vitro to examine the effects of MDSC induction. Gene ontology and KEGG pathway analyses were performed to analyze these differentially expressed genes. Reduced representation bisulfite sequencing was performed in TGF-?1-treated and untreated cells to evaluate differences in the methylation status and analyze the chromosomal distribution of differentially methylated sites (DMSs). Significant morphological changes to cells were observed at PP3, and most PP3 cells were positive for desmin and SCA-1, and were confirmed to be MDSCs. Results of western blot and immunohistochemistry analyses suggested that expressions of a-SMA and CNN1 significantly increased after treatment with TGF-?1. Global transcriptome analysis identified 1996 differentially expressed genes (MSC_TGF?1/MSC_NC). Results of methylome analysis indicated that there were more hypermethylation sites in the untreated group than in the TGF-?1-treated group. Most DMSs were hypermethylated, whereas a small portion was hypomethylated. The chromosomal distribution of DMSs indicated that chromosome 1 had the highest proportion of DMSs, whereas the Y chromosome had the fewest DMSs. Sud2, Pcdh19, and Nat14 are potential core genes involved in cell differentiation. These results may explain the mechanisms of cell differentiation and provide useful information regarding diseases such as pelvic organ prolapse.

SUBMITTER: Tang X

PROVIDER: S-EPMC6731421 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Molecular mechanisms involved in TGF-β1-induced Muscle-derived stem cells differentiation to smooth muscle cells.

Tang Xiang X Su Xianghui X Zhong Zhuohui Z Wen Canliang C Zhang Tiansong T Zhu Yali Y

American journal of translational research 20190815 8

We investigated the molecular mechanisms involved in transforming growth factor beta 1 (TGF-β1)-induced myogenic stem cell differentiation to smooth muscle cells. We isolated muscle-derived stem cells (MDSCs) from gastrocnemius muscles following their identification by immunohistochemistry analysis of desmin and flow cytometry analysis of SCA-1, CD34, and CD45. MDSCs at passage 3 (PP3) were cultured <i>in vitro</i> to examine the effects of MDSC induction. Gene ontology and KEGG pathway analyses ...[more]

PMID: 31497230

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Project description:BackgroundThe vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R.MethodsPrimary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs.FindingsThe mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-β1, TGF-β pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-β1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-β1 levels.InterpretationIn an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-β1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R.FundingNational Institute on Aging at the National Institutes of Health grant number R01AG064420.Research in contextEvidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-β1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-β1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-β1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.

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Molecular mechanisms involved in TGF-?1-induced Muscle-derived stem cells differentiation to smooth muscle cells.

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Molecular mechanisms involved in TGF-β1-induced Muscle-derived stem cells differentiation to smooth muscle cells.

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