Abstract
Purpose :
Pathological angiogenesis is a key factor in numerous ocular diseases. Recent work from our lab has demonstrated that one’s genetic background influences the responsiveness to angiogenic growth factors. In this study, the genetic diversity of common inbred mouse strains was utilized to identify novel candidate genes responsible for differences in angiogenic response through GWAS.
Methods :
The corneal neovascularization assay was performed on 39 different inbred mouse strains using 200 ng VEGFA pellets. After seven days, the total blood vessel area was analyzed in AngioTool. Efficient mixed-model association (EMMA) was used to identify loci in the genome that were associated with the observed variation in blood vessel area. The role of candidate genes situated in those loci was further evaluated using gene knockdown by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells (HUVECs). The extent of gene knockdown was checked by western blotting. The effect of the gene knockdown on angiogenesis was examined by in vitro cell proliferation and cell migration assays.
Results :
Data analysis by EMMA yielded four peaks with genome-wide significance on chromosomes 2, 5, 11, and 16. Six genes were prioritized by examining the available sequence of each gene for variants that may alter the amino acid sequence. One of them was located on chromosome 2 and the rest were located on chromosome 11. SiRNA knockdown and in vitro functional assays were used to determine the role of these genes in angiogenesis. Western blotting revealed ~91% knockdown of PYCR1 by siRNA. In vitro assays on HUVECs treated with PYCR1 siRNA revealed a 53% decrease in cell proliferation at day 3 as compared to scrambled siRNA treated HUVECs. However, PYCR1 siRNA treated HUVECs did not show any significant difference on cell migration as compared to scrambled siRNA treated HUVECs.
Conclusions :
The preliminary data has shown PYCR1 as a novel candidate gene that could affect angiogenic responsiveness. PYCR1 encodes Pyrroline-5-carboxylate reductase 1, which catalyzes the conversion of pyrroline-5-carboxylate to proline, which supports cancer cell proliferation and survival in oxygen-limiting conditions. However, more in vitro and in vivo assays will be performed to confirm this finding. These studies will aid our understanding of angiogenesis in growing, damaged, ischemic, or cancerous tissues.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.