Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling in endothelial precursor cells of patients protected from microvascular complications

ABSTRACT: In this study, we examined the impact of modulating the TGF-β/PAI-1 axis in CD34+ cells function from diabetic patients and controls. Using gene array studies, we found that diabetics, protected from microvascular complications despite suboptimal glycemic control, had reduced level of TGF- β1 and PAI-1 transcripts in their CD34+ cells compared to age, sex, duration and degree of glycemic control -matched diabetics with microvascular complications. Treatment with neutralizing antibody to TGF- β1 in murine hematopoietic stem cells (HSC) enhanced in vivo repopulation potential of these cells in bone marrow transplantation; reduced the time required for cell division of single cells, increased survival of the progenitor cells and reduced TGF-β1 expression. TGF- β1 phosphorodiamidate morpholino oligomers (PMO) treatment reduced PAI-1 mRNA expression in diabetic (p<0.01) and non-diabetic (p=0.05) CD34+ cells. Inhibition of PAI-1 promoted CD34+ cell proliferation and migration in vitro.Targetting TGFβ-1/PAI-1 system offers a promising therapeutic strategy for restoring vascular reparative function in diabetic CD34+ cells and hematopoietic stem cells, enhancing key functions needed for cell therapy. Peripheral blood from either age matched controls (n=5), diabetics with long standing poorly controlled diabetes and no microvascular complications (n=4), and diabetics with long-standing poorly controlled diabetes with severe microvascular complications (n=5) was obtained and CD34+ cells were isolated. RNA was extracted followed by AffyNugen amplification, and the cDNA was probed to Human RSTA Affymetrix 2.0 chip

ORGANISM(S): Homo sapiens

SUBMITTER: Ashay Bhatwadekar

PROVIDER: E-GEOD-43950 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Microarray analysis reveals up-regulation of retinoic acid and hepatocyte growth factor related signaling pathways by pro-insulin C-peptide in kidney proximal tubular cells: Antagonism of the pro-fibrotic effects of TGF-b1 Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or transforming growth factor beta 1 (TGF-β1) in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor β (RARβ), hepatcoyte growth factor (HGF), cellular retinoic acid binding protein II (CRABPII), vimentin, E-cadherin, Snail and β-catenin was assessed by immunoblotting. The cellular localisation of vimentin and β-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1,458 genes after C-peptide exposure for 18h or 48h respectively. From these, members of the anti-fibrotic retinoic acid (RA) and HGF signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARβ, CRABPII and HGF. We confirmed a role for RA in reversal of TGF-β1-induced changes associated with epithelial-mesenchymal transition (EMT), including expression changes in Snail, E-cadherin, vimetin and redistribution of β-catenin. Importantly, these TGF-β1-induced changes were inhibited by C-peptide. Further, effects of TGF-β1 on Snail and E-cadherin expression were blocked by HGF and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.

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Gene expression profiling in endothelial precursor cells of patients protected from microvascular complications

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