PADI2-mediated citrullination regulates the differences in angiogenic response

Abstract

Purpose : We have recently identified Padi2 (peptidyl arginine deiminase type II) as a novel angiogenesis-regulating gene by genome-wide association in common inbred mice strains. Here, we further defined the role of PADI2 in angiogenesis and developed both a knock-out (KO) mouse strain and a padi2 null zebrafish in the kdrl:zsGreen transgenic line.

Methods : In order to inactivate Padi2 gene, we used the CRISPR-Cas9 system where we designed specific gRNAs against the conserved PADI catalytic domain specific to Padi2 gene in 129S1/SvImJ strain. To determine whether PADI2 modulates angiogenic response in mice, we used the corneal micropocket neovascularization assay with 20ng of bFGF. To generate a mutant padi2 line in zebrafish, we made small deletions within the catalytic domain of padi2 gene also using CRISPR-Cas9 system in the kdrl:zsGreen transgenic zebrafish. Vascular defects were evaluated in mutant lines within 72 hours post fertilization (hpf). Citrullination levels in both zebrafish and mouse cornea were evaluated by western blot.

Results : Excitingly, we observed a significant decrease of angiogenic response in homozygous Padi2 KO compared to wild type control mice. This corresponded with a significant decrease in citrullination levels in Padi2 KO cornea compared to wild type controls. In zebrafish, wild type and heterozygous mutant fish did not show any vascular defects while significant vascular defects were observed in homozygous padi2 null embryos. We confirmed the padi2 null alleles in both mice and zebrafish by Western blot. Furthermore, off-springs from heterozygous incrosses of different zebrafish padi2 mutant alleles were genotyped at 72 hpf; in total 244 fish were analyzed. Compared with a Mendelian ratio, live padi2 null fish were underrepresented in clutches of all heterozygous crosses. Our data suggests a reduced survival of homozygous padi2 mutants.

Conclusions : Our results demonstrate, for the first time, the key role of Padi2 as an angiogenesis-regulating gene. Furthermore, the severe vascular phenotype in homozygous padi2 mutant fish and a significant decrease in angiogenic response in Padi2 KO mice strongly support our hypothesis that PADI2-mediated citrullination play a key role in regulation of angiogenic response. Characterization of PADI2 and others in the associated pathways can reveal novel therapeutic targets for a wide variety of angiogenesis-dependent diseases in ophthalmology and beyond.

This is a 2020 ARVO Annual Meeting abstract.