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.

Fig 1. Angiogenic response in the cornea of common inbred mouse strains.

Mean ± SD for measured vessel area using slow release 20 ng of bFGF pellets in cornea of 42 common inbred mouse strains. The difference between the strains with the lowest and the highest values were 4.88-fold. The cornea micropocket eye assay was not possible in PL/J mice due to proptosis.

Fig 2. GWAS results for vessel area using EMMA.

Manhattan plot showing the EMMA adjusted association (-log10) p-values (-logP) for vessel area in 42 common inbred mouse strains. The analysis was performed using 132k SNPs with a minor allele frequency > 5%. Each chromosome is plotted on the x-axis. SNPs on Chr. 4 (rs32857122; p = 9.34 x 10⁻⁷ and rs32259427; 3.67 x 10⁻⁶) show GWAS peaks with highest significance.

Fig 3. Gene expression of a candidate gene within the most statistically significant region identified by EMMA.

Expression data of Padi2 show a strong correlation with haplotypes among inbred mice who have “low” or “high” angiogenic responses. Coordinating colors represent inbred mice who share the same haplotype. Data represent mean ± standard error of the mean (SEM) from 8 age-matched corneas. * and ** indicates P < 0.01 and P < 0.001 respectively.

Fig 4. Regional plot of the Chr. 4 association in 42 common inbred mouse strains centered on the lead SNP (rs32857122) at the 5’-untranslated region of Padi2 locus.

The top two black dots represents the most significant SNPs (p = 9.34 x 10⁻⁷ and 3.67 x 10⁻⁶ respectively). The positions of all reference sequence genes are plotted using genome locations from UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly.

Fig 5. Padi2 expression level corresponds with the level of citrullinated protein in both unstimulated and bFGF stimulated corneas.

A) Western blot of unstimulated cornea from C57BL/6J and 129S1/SvImJ verifying the RT-PCR experiments. B) Immunofluorescent staining of unstimulated corneas from C57BL/6J and 129S1/SvImJ. Quantification of Padi2 staining relative to cell number (S5 Fig) is higher, as visible from the green staining, at the limbal basal epithelial cells and limbal vasculatures of cornea from 129S1/SvImJ strain. C) Co-staining experiment for Abcg2 and Padi2 using confocal laser scanning microscopy of bFGF stimulated corneas from C57BL/6J and 129S1/SvImJ. Padi2 is clearly expressed in both Abcg2⁺ limbal basal cells and new bFGF-induced vessels. Scale bar = 100 μm. D) Knockdown of PADI2 and inhibition by Cl-Amidine treatment significantly reduces deiminated proteins in HMVECs. Significant difference in intrapeptide citrullination between the two strains. Anti-Citrulline antibody (Abcam, Cambridge, MA) used in this experiment does not react with free citrulline or arginine and reacts only with intrapeptidic citrulline regardless of the amino acid sequence. We further verified this by HPLC (S5 Fig).

Fig 6. In vitro endothelial cell functional assay.

A) HMVEC migration in response to full serum media (FSM) was significantly decreased when cells were transfected with PADI2-specific siRNA compared to scramble siRNA control. Started with seeded 20,000 cells per well; Basal medium containing 0.1% BSA is used as negative control. We did not find any difference in endothelial cell proliferation (S5 Fig). B) HMVEC migration in response to FSM was significantly decreased upon treatment with 1mM Cl-Amidine, a PADI inhibitor, compared to DMSO treated control. We observed no effect in 3T3 fibroblast cell migration when treated with Cl-Amidine. * indicates P < 0.05. † Treatment with 10 mM Cl-Amidine was toxic to both cell lines. C) HMVEC migration and proliferation (D) was assessed when human PADI2 is overexpressed. We observed a significant increase in both endothelial cells migration and proliferation (D) when PADI2 is overexpressed compared to vector only transfected cells. Started with seeded 10,000 cells per well. * and ** indicates P < 0.05 and P < 0.001 respectively.

Fig 7. padi2 morphants display vascular defects at 48 hpf.

A) Significant vascular defects in padi2 MO compared to sham-injected MO (negative) control siblings (representative figures from 16 injections, repeated 4 times). Note the gaps present in the intersegmental vessels suggesting missing or abnormal formation. Also note vessel absence in the head (arrows). Magnification: x10. B) Higher magnificantion imgages showing head vascular defects in embryos injected with zebrafish padi2 MO.

Fig 8. Human PADI2 mRNA can rescue vessel defects in zebrafish padi2 morphants.

A) Significant vascular defects (arrows) are present in padi2 MO (1.5 ng/embryo) (b) compared to standard (sham-injected) MO control (1.5 ng/embryo) (a) and human PADI2 (hPADI2) mRNA only (225 pg/embryo) (c). Co-injection of hPADI2 mRNA (225 pg/embryo) rescues padi2 MO (1.5 ng) vascular defect. (d) Magnification: x10. B) Bar graph of normal and defective ISVs in four different groups, showing average ±SD from four independent experiments. Missing and abnormal formation of ISVs are considered as defective vessels. The number of defective vessels is significantly increased with the injection of padi2 MO (* and ** denotes P<0.001 and P<0.005 respectively.) C) Citrullination levels in zebrafish. The level of deiminated protein is partially rescued in padi2 morphants with the co-injection of hPADI2 mRNA.