Abstract
Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.
Highlights
Angiogenesis, the process by which new blood vessels are formed from existing vessels, plays a key role in a number of human diseases such as cancer, rheumatoid arthritis, cardiovascular disease, diabetic retinopathy and macular degeneration
Angiogenesis plays a key role in a number of human diseases such as cancer, rheumatoid arthritis, cardiovascular disease, and macular degeneration
We have used the genetic diversity available in common inbred mouse strains to identify quantitative trait loci (QTLs) responsible for differences in angiogenic response and further refined the mapping of a genome-wide significant peak on chromosome 4. We used both expression analyses and zebrafish model to successfully identify peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, as the gene responsible for a significant portion of the difference in angiogenic responsiveness. These data are the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene and opens potential therapeutic avenues for a wide variety of systemic angiogenesis-dependent diseases
Summary
Angiogenesis, the process by which new blood vessels are formed from existing vessels, plays a key role in a number of human diseases such as cancer, rheumatoid arthritis, cardiovascular disease, diabetic retinopathy and macular degeneration. Regulation of angiogenesis is determined by a balance of pro- and anti- angiogenic signals and their interaction with endothelial cells and surrounding stroma. Important angiogenic regulators include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietins (ANG1, ANG2) and platelet derived growth factor (PDGF). BFGF is an extracellular matrix-bound protein normally released during wound healing. VEGF activity is regulated by the expression of different isoforms and binding to different VEGF receptors and inhibitors [5]. It signals through binding with fibroblast growth factor receptors (FGFR), a family of membrane receptors with tyrosine kinase activity [6]. Endogenous inhibitors of angiogenesis include soluble VEGF receptors, neuropilins, angiostatin, and thrombospondins, all of which are important in both health and disease
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