Ventroptin, a BMP4 Antagonist, Is Upregulated by Hypoxia in Retinal Pericytes

Abstract

Abstract: : Purpose: Diabetes–specific microvascular disease of the retina is characterised by abnormalities in blood flow and increased vascular permeability. Abnormal angiogenesis in diabetes is most clinically apparent in proliferative diabetic retinopathy. Occlusion of small retinal capillaries results in localised ischemia, leading to increased retinal permeability and neovascularisation. Retinal pericytes play a role in the regulation of angiogenesis and neovascularisation by providing vascular stability and controlling endothelial cell proliferation. We have investigated differential gene expression in an in vitro model of diabetic retinopathy: a culture of human retinal pericytes exposed to normoxic and hypoxic (1% O₂) conditions. Methods: Human retinal pericytes were cultured in MCDB 131, 5% FBS, 2mM L–glutamine, 100U/ml penicillin and 0.1mg/ml streptomycin. Cells, once confluent, were cultured in hypoxic (1% O₂) conditions for 48 hours, with normoxic conditions as control. Affymetrix gene chip analysis was carried out. Genes upregulated greater then 1.5 fold in each of three replicate experiments were considered for further study. Gene promoters were obtained from the Human Genome Browser Gateway (http://genome.ucsc.edu/). Potential transcription factor binding sites were investigated using MatInspector software (http://www.genomatix.de). Results: There were 28 genes upregulated and 7 genes downregulated 1.5 fold or greater. Analysis of the promoters of the upregulated genes demonstrates common transcriptional regulatory motifs. Common to many of the promoters are binding motifs for the transcription factors HIF–1α and CREB. We demonstrate that overexpression of HIF–1α in retinal pericytes induces increased mRNA expression of known HIF–1α responsive genes, such as VEGF and Cox–2. We also observe increases in expression of mRNA encoding the BMP4 antagonist Ventroptin. This is a novel gene in the context of diabetic retinopathy. We have expressed recombinant Ventroptin in pericytes and report that secreted Ventroptin binds to BMP4. Conclusions: Hypoxia upregulated genes in retinal pericytes could be classified into functional groups, including, growth factors, angiogenesis, transcriptional regulation and developmental. Expression of Ventroptin, a novel gene in the context of diabetic retinopathy, in response to hypoxia may modulate the effects of BMP4 on the retinal endothelium.

Keywords: diabetic retinopathy • hypoxia • gene microarray