Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice

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.

Applicability of Precision Medicine Approaches to Managing Hypertension in Rural Populations

“…The eSNPs we identified associated with a robust change in PADI2 expression in multiple tissues including blood cells, heart, aorta, and both visceral and subcutaneous adipose ( Table S9). Altered PADI2 activity is implicated in neurodegenerative and inflammatory diseases [54] and most recently, vascular angiogenesis [55].…”

Identification of Basp1 as a novel angiogenesis‐regulating gene by multi‐model system studies

“…Total RNA was extracted from corneas as previously described. 7 Briefly, corneas from five mice per group were harvested and immediately stored in RNAlater RNA Stabilization reagent (Qiagen). Each cornea was trimmed excluding the limbus and was homogenized using PRO200 homogenizer (PRO Scientific).…”

“…We previously identified two different genome-wide significant association peaks on Chromosome 15, one is located at 24 682 428 bp (SNP rs33886061) and another at 96 431 470 bp (SNP rs50093952). 7 Within each association peak, we selected candidate genes that either were coding nonsynonymous SNPs with potential functional consequences or had SNPs that were present within the promoter or untranslated regions that could alter the gene expression. We previously identified, Slc38A1 (Solute Carrier Family 38, Member 1, PMID: 608 490) that bears a coding-nonsynonymous SNP (rs50093952) in an amino acid residue that is conserved in all mammalian species.…”

“…We successfully identified Basp1, brain abundant membrane attached signal protein 1, as a major contributing gene in the regulation of angiogenesis among the strains that were phenotyped in our assay. 7 BASP1 (also known as NAP22) was originally identified in brain extracts and it is particularly abundant in nerve terminals during brain development and it is known to be involved in axon regeneration. 10,11 BASP1 is also expressed in various other tissues, 12,13 but its precise functions are still unknown.…”

Applicability of Precision Medicine Approaches to Managing Hypertension in Rural Populations

“…The eSNPs we identified associated with a robust change in PADI2 expression in multiple tissues including blood cells, heart, aorta, and both visceral and subcutaneous adipose ( Table S9 ). Altered PADI2 activity is implicated in neurodegenerative and inflammatory diseases [ 54 ] and most recently, vascular angiogenesis [ 55 ]. PADI2 expression and anti-citrullinated protein antibodies were higher in smokers, suggesting that increased PADI2 expression, particularly in genetically susceptible subjects ( Figure 7 b), promotes more robust pathophysiological responses to environmental stressors [ 56 , 57 , 58 ].…”