505 Epigenetic Regulation of Inflammation-Induced Endothelial-to-Mesenchymal Transition (EndoMT): A Novel Mechanism of Fibrosis in Inflammatory Bowel Disease (IBD)

Abstract

BACKGROUND: Intestinal fibrosis is a common and potentially serious complication of inflammatory bowel disease (IBD). In addition to mesenchymal cells, endothelial cells can contribute to fibrosis through the recently described process of EndoMT. We investigated whether i) human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT and contribute to fibrosis in IBD, and ii) epigenetic changes are involved in mediation of EndoMT.MATERIALS ANDMETHODS: HIMEC exposed to TGF-β1, IL-1β and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) were evaluated for morphological, phenotypic and functional changes compatible with EndoMT. Microarray genomic analysis was used to identify transcription factors controlling the transformation process. Evidence of in situ and In Vivo EndoMT was sought in human and experimental IBD tissues. Chromatin immunoprecipitation (ChIP) assays were performed to identify specific histone modification changes on the promoters of von Willebrand factor (vWF) and collagen1 alpha2 (Col1α2) genes (markers of endothelial cells and fibroblasts, respectively) during EndoMT. RESULTS: The combination of TGF-β1, IL-1β and TNF-α induced morphologic and phenotypic changes in HIMEC consistent with EndoMT, and these were reproduced using supernatants of activated LPMC. Transformed HIMEC exhibited loss of acetylated LDL-uptake, enhanced extracellular matrix secretion, and acquisition of de novo collagen synthesis capacity. These functional alterations persisted at 10 days after removal of the inducing agents, and then still were associated with an approximate 8-fold upregulation of Col1α2 gene expression and 1.6-fold downregulation of vWF gene expression. EndoMT was detected in inflamed microvessels of human IBD mucosa and TNBS-induced colonic fibrosis of Tie2-GFP reporter mice. Genomic analysis uncovered Sp1 as the dominant transcriptional regulator of EndoMT. Finally, ChIP analysis revealed increased methylation of lysines 9 and 27 in histone H3 associated with repression of vWF gene expression, while increased acetylation of histone H4 accompanied activation of Col1α2 gene expression. CONCLUSIONS: Inflammatory stimuli induce trans-differentiation of mucosal microvascular cells into mesenchymal cells In Vitro and In Vivo, supporting the notion that the microvasculature undergoes EndoMT in IBD and contributes to fibrogenesis. These events involve epigenetic changes such as histone modifications crucial to regulation of EndoMT, and define unique pro-fibrotic epigenotypes that could become targets of selective anti-fibrotic therapies in IBD.