A functional study of blood-pressure-associated SNPs at natriuretic peptide receptor C gene locus.
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Background: Essential hypertension is regarded as a complex disease, the phenotype of which results from interactions between numerous genes and environmental factors. Genome-wide association studies of blood pressure (BP) and hypertension have been developed to explore the potential genes involved in blood pressure and identified a number of trait-associated variants. Among those variants, single nucleotide polymorphisms (SNPs) rs1173771 (G/A) and rs1421811 (G/C) are located at the natriuretic peptide receptor C (NPR3) gene locus. Their major alleles are related with blood pressure elevation. Studies have implicated NPR-C in mediating some of the cardio-protective actions of natriuretic peptides and its direct involvement in the pathogenesis of hypertension. However, the precise role of these association between genetic variants at NPR3 and blood pressure control has not been elucidated. Objective: To functionally characterise the effect of BP-associated SNPs at the NPR3 gene locus in the context of BP regulatory pathways. Methods: Primary human umbilical artery smooth muscle (HUASMCs) and vein endothelial (HUVECs) cells were genotyped for BP-associated NPR3 variants. Endogenous mRNA and protein expression levels were assessed by qRT-PCR, allelic expression imbalance assay and western blotting. Open chromatin regions were assayed using formaldehyde-assisted isolation of regulatory elements (FAIRE). Interaction between variants flanking region with nuclear protein was detected by electrophoretic mobility shift assay (EMSA). Cell proliferation and migration were 4 determined by cell counting and scratch assays. Angiotensin II (Ang II)-induced calcium flux was evaluated using the intracellular fluorescent probe. Results: The BP-elevating allele of the NPR3 variants in rs1173771 linkage disequilibrium (LD) block was associated with lower endogenous mRNA and protein levels in HUASMCs. This is consistent with the finding that BP-elevating allele is less located within open chromatin. The decreased NPR3 expression in HUASMCs carrying the BP-elevating allele is associated with increased cell proliferation and intracellular calcium flux in response to Ang II stimulation. No differences in migration rates were detected. No genotype-dependent characteristics were observed in HUVECs NPR3 expression and cell proliferation. Moreover, RT-PCR showed a linkage between of the BP-elevating allele of the NPR3 variants in rs1421811 LD block and lower endogenous mRNA in HUASMCs. Intracellular calcium flux detection also revealed a trend of higher response to Ang II stimulation in BP-elevating allele homozygous HUASMCs. However, No genetic differences were detected in proliferation and migration rates of HUASMCs, and HUVECs NPR3 expression and cell proliferation studies did not present any significant genotype-dependent association. Conclusions: This study has identified a potential mechanism for BP-associated SNPs at NPR3 locus to influence BP predominantly via an effect on vascular smooth muscle cell behaviours.
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