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Mehrdad Khajavi, Yi Zhou, Amy Birsner, Lauren Bazinet, Bella Hu, Leonard Zon, Robert J D'Amato; Identification of novel angiogenesis-regulating genes by genome association studies in mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4345.
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© ARVO (1962-2015); The Authors (2016-present)
Recent findings indicate that stimulus-driven angiogenesis is controlled by genetic variation. This difference in angiogenic responsiveness may alter the susceptibility to a number of blinding angiogenesis-dependent diseases. The goal of our study is to elucidate the genetic basis of differences in angiogenic responsiveness.
We utilized the genetic diversity available in common inbred mouse strains to identify loci responsible for differences in angiogenic response. Corneal neovascularization assay was performed on 40 different inbred mouse strains using 20 ng bFGF pellets. We next employed efﬁcient mixed-model association (EMMA) mapping using the vessel area data from all strains. Expression analyses of candidate genes in corneas of 5 different inbred strains with various angiogenic responses were performed using quantitative RT-PCR. We further evaluated their role in angiogenesis by in vitro functional assays in human microvascular endothelial cells (HMVEC) and in vivo functional modeling in zebrafish embryos.
Our preliminary analysis yielded five peaks with genome-wide significance on chromosome 4, 5, 11, 15 and 16. We identified 7 candidate genes by examining the available sequence of each gene for variants that may alter amino acid sequence or gene expression. Of these, one gene (Irf2bp2) was already implicated in the regulation of angiogenesis, serving as a confirmation of our approach. The other candidate genes were novel and known to be involved in metabolic, signaling and structural pathways. Our expression data from 4 out of 5 genes showed a strong correlation with regions containing shared haplotype, suggesting the existence of an eQTL at these loci. In addition, knockdown of each of the 5 candidate genes by siRNA in HMVECs led to significant decreases in migration. Furthermore, morpholino knockdown in zebrafish embryos demonstrated altered vascular patterns in morphants of 3 out of 5 candidate genes compared to wild-type control siblings.
Our genetic screen has identified 5 candidate genes in various pathways that could affect angiogenic responsiveness. The results suggest that characterization of these genes and others in associated pathways may provide valuable new information and new therapeutic targets useful for a wide variety of angiogenesis-dependent diseases.
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