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Analysis of Gene Expression changes in human intestinal endothelial-to-mesenchymal transition - TRANSFORMED GROUP

ABSTRACT: Intestinal fibrosis is a well-known complication of inflammatory bowel disease (IBD) and has important clinical implications for both forms of IBD, Crohn's disease (CD) and ulcerative colitis (UC). In addition to mesenchymal cells, endothelial cell contribute to organ fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). Our aim was to investigate if human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT, produce extracellular matrix, and contribute to fibrosis in both forms of IBD. Cellular transformation is a highly complex event involving drastic changes in multiple gene expression levels. To assess global genomic changes accompanying HIMEC transformation, we performed microarray analysis comparing transformed to non-transformed/untreated HIMEC. Consistent with the morphological, phenotypic and functional findings, transformed HIMEC downregulated the expression levels of genes typically expressed in endothelial cells and upregulated several genes of ECM molecules known to be increased in intestinal fibrosis. Thus, it is evident that inflammatory stimuli induce transdifferentiation of HIMEC into matrix-producing mesenchymal cells. Because the conditions inducing transformation in vitro reproduce those found in vivo during active gut inflammation, the microvasculature may contribute to IBD-associated fibrosis through the novel process of EndoMT. This series includes all treated or TRANSFORMED HIMEC samples (4).

ORGANISM(S): Homo sapiens

PROVIDER: GSE15934 | GEO | 2011/11/02

SECONDARY ACCESSION(S): PRJNA123047

REPOSITORIES: GEO

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Project description:Intestinal fibrosis is a well-known complication of inflammatory bowel disease (IBD) and has important clinical implications for both forms of IBD, Crohn's disease (CD) and ulcerative colitis (UC). In addition to mesenchymal cells, endothelial cell contribute to organ fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). Our aim was to investigate if human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT, produce extracellular matrix, and contribute to fibrosis in both forms of IBD. Cellular transformation is a highly complex event involving drastic changes in multiple gene expression levels. To assess global genomic changes accompanying HIMEC transformation, we performed microarray analysis comparing transformed to non-transformed/untreated HIMEC. Consistent with the morphological, phenotypic and functional findings, transformed HIMEC downregulated the expression levels of genes typically expressed in endothelial cells and upregulated several genes of ECM molecules known to be increased in intestinal fibrosis. Thus, it is evident that inflammatory stimuli induce transdifferentiation of HIMEC into matrix-producing mesenchymal cells. Because the conditions inducing transformation in vitro reproduce those found in vivo during active gut inflammation, the microvasculature may contribute to IBD-associated fibrosis through the novel process of EndoMT. This series includes all treated or TRANSFORMED HIMEC samples (4). Surgically resected specimens were used for isolation of human intestinal microvascular endothelial cells (HIMEC). HIMEC monolayers derived from intestinal mucosa of four different subjects – 2 controls, 1 ulcerative colitis, and 1 crohns were cultured in 75cm2 tissue culture flasks coated with fibronectin. Cells were left untreated (unstimulated/non-transformed) or exposed to the combination of IL-1b 100 U/ml, TNF-a 100 U/ml and TGF-b1, 5ng/ml (TGFTNFIL1) for six days. HIMEC medium was changed every 3 days. Total ribonucleic acid (RNA) was extracted using a commercially available kit (Qiagen®). RNA was reverse transcribed into cRNA which was then hybridized to the Illumina HumanRef-8 v2 Expression BeadChip. The raw gene expression datasets were processed to remove outliers, log2 transformed and quantile normalized using R lumi software. Those genes that satisfied the FDR p-value threshold of < 0.05 or raw p-value of <0.001 were identified as significant for the functional pathway and network analysis.

Project description:Intestinal fibrosis is a well-known complication of inflammatory bowel disease (IBD) and has important clinical implications for both forms of IBD, Crohn's disease (CD) and ulcerative colitis (UC). In addition to mesenchymal cells, endothelial cell contribute to organ fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). Our aim was to investigate if human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT, produce extracellular matrix, and contribute to fibrosis in both forms of IBD. Cellular transformation is a highly complex event involving drastic changes in multiple gene expression levels. To assess global genomic changes accompanying HIMEC transformation, we performed microarray analysis comparing transformed to non-transformed/untreated HIMEC. Consistent with the morphological, phenotypic and functional findings, transformed HIMEC downregulated the expression levels of genes typically expressed in endothelial cells and upregulated several genes of ECM molecules known to be increased in intestinal fibrosis. Thus, it is evident that inflammatory stimuli induce transdifferentiation of HIMEC into matrix-producing mesenchymal cells. Because the conditions inducing transformation in vitro reproduce those found in vivo during active gut inflammation, the microvasculature may contribute to IBD-associated fibrosis through the novel process of EndoMT. This series includes all untreated or CONTROL HIMEC samples (4). Surgically resected specimens were used for isolation of human intestinal microvascular endothelial cells (HIMEC). HIMEC monolayers derived from intestinal mucosa of four different subjects – 2 controls, 1 ulcerative colitis, and 1 crohns were cultured in 75cm2 tissue culture flasks coated with fibronectin. Cells were left untreated (unstimulated/non-transformed) or exposed to the combination of IL-1b 100 U/ml, TNF-a 100 U/ml and TGF-b1, 5ng/ml (TGFTNFIL1) for six days. HIMEC medium was changed every 3 days. Total ribonucleic acid (RNA) was extracted using a commercially available kit (Qiagen®). RNA was reverse transcribed into cRNA which was then hybridized to the Illumina HumanRef-8 v2 Expression BeadChip. The raw gene expression datasets were processed to remove outliers, log2 transformed and quantile normalized using R lumi software. Those genes that satisfied the FDR p-value threshold of < 0.05 or raw p-value of <0.001 were identified as significant for the functional pathway and network analysis.

Project description:Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive an endothelial-to-mesenchymal transition (EndoMT), where endothelial cells undergo a phenotypic switch to mesenchymal-like cells that are pro-inflammatory and pro-fibrotic. In this study, we sought to determine whether obesity can promote EndoMT in adipose tissue. Mice in which endothelial cells are lineage-traced with eYFP were fed a high-fat/high-sucrose (HF/HS) or Control diet for 13, 26, and 52 weeks, and EndoMT was assessed in adipose tissue depots as percentage of CD45-CD31-Acta2+ mesenchymal-like cells that were eYFP+. EndoMT was also assessed in human adipose endothelial cells through cell culture assays and by the analysis of single cell RNA sequencing datasets obtained from the visceral adipose tissues of obese individuals. Quantification by flow cytometry showed that mice fed a HF/HS diet display a time-dependent increase in EndoMT over Control diet in subcutaneous adipose tissue (+3.0%, +2.6-fold at 13 weeks; +10.6%, +3.2-fold at 26 weeks; +11.8%, +2.9-fold at 52 weeks) and visceral adipose tissue (+5.5%, +2.3-fold at 13 weeks; +20.7%, +4.3-fold at 26 weeks; +25.7%, +4.8-fold at 52 weeks). Transcriptomic analysis revealed that EndoMT cells in visceral adipose tissue have enriched expression of genes associated with inflammatory and TGFb signaling pathways. Human adipose-derived microvascular endothelial cells cultured with TGF-β1, IFN-γ, and TNF-⍺ exhibited a similar upregulation of EndoMT markers and induction of inflammatory response pathways. Analysis of single cell RNA sequencing datasets from visceral adipose tissue of obese patients revealed a nascent EndoMT sub-cluster of endothelial cells with reduced PECAM1 and increased ACTA2 expression, which was also enriched for inflammatory signaling genes and other genes associated with EndoMT. These experimental and clinical findings show that chronic obesity can accelerate EndoMT in adipose tissue. We speculate that EndoMT is a feature of adipose tissue dysfunction that contributes to local inflammation and the systemic metabolic effects of obesity.

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Analysis of Gene Expression changes in human intestinal endothelial-to-mesenchymal transition - TRANSFORMED GROUP

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