Abstract P048: Blood-Pressure Associated Variants in Natriuretic Peptide Receptor C Affect Human Vascular Smooth Muscle Cells Proliferation and Calcium Flux in Response to Angiotensin II

Abstract

Introduction: A recent genome-wide association study revealed a significant association between variation at natriuretic peptide receptor C (NPR3) gene locus and blood pressure (BP). Objective: To functionally characterise the effect of BP- associated SNPs (single nucleotide polymorphisms) at the NPR3 gene locus in the context of BP regulatory pathways. Methods: A collection of primary human umbilical smooth muscle (HUASMCs) and endothelial (HUVECs) cells were genotyped for NPR3 gene SNP rs1173743, rs1173747, rs1173756 and rs1173771. Endogenous mRNA and protein expression levels were assessed by qRT-PCR and western blotting. Open chromatin regions were assayed using formaldehyde-assisted isolation of regulatory elements. Cell proliferation and migration were detected by cell counting and scratch assays. Angiotensin II (AngII)-induced Calcium flux was investigated using an intracellular fluorescent probe (Calcium 6). Results: The NPR3 gene risk allele (major allele) of intronic SNP rs1173747 was associated with lower endogenous mRNA (p<0.001) and protein levels in HUASMCs. This was consistent with its minor allele being located within an open chromatin state (p<0.05). Furthermore, cells carrying the major-allele of rs1173747 had increased proliferation (p<0.05) and Calcium flux in response to AngII stimulation (p<0.05). No difference in migration rates were detected. No such genotype-dependent characteristics were observed in HUVECs. Conclusions: This study has identified potential mechanisms for BP-associated SNPs in NPR3 gene locus to influence BP predominantly via effect on vascular smooth muscle cell behaviours