The biological impact of blood pressure-associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle

Figure 1.. Regional plots for association of NPR3 variants with SBP/DBP/PP within the UK Biobank cohort. (A–C) Genome-wide primary association analysis of main effects for medication-adjusted SBP, DBP and PP traits using the UK Biobank cohort (N=140 886) showed two signals at the NPR3 locus. The sentinel SNPs in these two independent linkage disequilibrium (LD) blocks are labelled in red or light blue text, respectively. The purple dot indicates rs1173771, the lead SNP in our previous GWAS study (2), and r2 measures of LD are shown in relation with rs1173771. (D) A conditional analysis based on the two published lead SNPs, rs1173771 and rs1421811, indicated the presence of a weak residual signal with rs1173747 in the rs1173771 LD block. Note that the variants (dark blue dots) upstream of rs1173747 were not significantly associated with BP in the primary main association analysis, so are not of interest within the conditional analysis.

Figure 2.. Blood pressure associated variants affect NPR3 expression in human VSMCs. For all panels, ‘E’ stands for the BP-elevating allele for each SNP, ‘L’ denotes the BP-lowering allele for each SNP. (A) Relative mRNA levels of NPR3 in VSMCs was determined by quantitative reverse-transcription polymerase chain reaction and standardized against the reference gene 18S. Mean±SEM, **P <0.01. (B) The relative peak heights of two alleles of the 3′ UTR SNP rs1173756 (r2=0.78 with lead SNP rs1173771) in sequencing chromatograms from genomic DNA and complementary DNA (cDNA) of VSMCs. Mean±SEM, **P <0.01. (C) A representative image of western blot analysis of NPR-C protein in VSMCs with β-actin as a loading control, Mean±SEM, *P <0.05.

Figure 3.. The BP-associated variant rs1173771 affects chromatin status and nuclear protein binding. In this figure, ‘E’ indicates BP-elevating allele, ‘L’ denotes BP-lowering allele. (A) Formaldehyde-assisted isolation of regulatory elements followed by allelic imbalance assay by Sanger sequencing. Left panel: Representative images showing different peak heights of the E and L alleles of rs1173771 in chromatograms derived from reference (Ref.) DNA and FAIRE DNA, respectively. Right panel: values in the bar chart are mean±SEM of n=6 heterozygous samples, *P <0.05. (B) Representative result of an electrophoretic mobility shift assay with the use of biotin-labelled double-stranded oligonucleotide probes and unlabelled competitors of the E and L alleles of rs11737711, and nuclear protein extracts from VSMCs. (C) Chromatin immunoprecipitation with an anti-H3K27ac antibody and IgG (the negative control) in heterozygous VSMCs. Upper left panel: PCR amplified DNA containing the rs1173771 site derived from anti-H3K27ac precipitated DNA, anti-H3K4me1 precipitated DNA and input DNA, respectively. Lower left panel: representative chromatographs showing the peak heights of the E and L alleles of…

Figure 4.. The BP-associated variant rs1173747 affects chromatin status and nuclear protein binding. In this figure, ‘E’ indicates BP-elevating allele, ‘L’ denotes BP-lowering allele. (A) Formaldehyde-assisted isolation of regulatory elements followed by allelic imbalance assays by Sanger sequencing. Representative images showing different peak heights of the E and L alleles of rs1173747 in chromatograms derived from reference (Ref.) DNA and FAIRE DNA. Mean±SEM of n=6 heterozygous samples, *P <0.05. (B) Representative result of electrophoretic mobility shift assay with the use of biotin-labelled double-stranded oligonucleotide probes and unlabelled competitors of the E and L alleles of rs1173747 and nuclear protein extracts from VSMCs. (C) Relative activity of the rs1173747 E and L alleles, respectively, in driving firefly luciferase reporter gene expression in transfected VSMCs, standardized for transfection efficiency. Mean±SEM, **P <0.01, ***P <0.001.

Figure 5.. The BP-elevating variant at NPR3 locus is associated with increased human VSMCs proliferation. VSMCs in the absence and presence of C-type natriuretic peptide (CNP; 100 nmol/l) and the NPR-C specific agonist (cANF4–23; 100 nmol/l) for 24 h were analysed for proliferation rate. Cell proliferation over the 24-h period was calculated as (OD450 at 24 h−OD450 at baseline)/OD450 at baseline. ‘E’ indicates the BP-elevating allele, ‘L’ denotes the BP-lowering allele. (A) Differences in proliferation between untreated cells of different genotypes. Representative images of DAPI stained cells of the three different genotypes at baseline and 24 h. Scale bars=200µm. Mean±SEM, *P <0.05. (B) Differences in proliferation between CNP or cANF4–23 treated cells of different genotypes, and (C) Inhibitory effects of CNP and cANF4–23 on proliferation of cells of different genotypes, calculated by (OD450 at 24 h of untreated cells−OD450 at 24 h of CNP-treated or cANF4–23-treated cells)/OD450 at 24 h of untreated cells. Mean±SEM, *P <0.05.

Figure 6.. The BP-elevating allele at NPR3 locus increases Ang II-induced intracellular calcium changes in human VSMCs. Human VSMCs were incubated with or without C-type natriuretic peptide (CNP; 1 μmol/l) and the NPR-C specific agonist cANF4–23(1 μmol/l), and then exposed to Ang II (10 or 100 nmol/l). ‘E’ indicates the BP-elevating allele, ‘L’ denotes the BP-lowering allele. (A) Representative images of intracellular calcium changes among genotypes under stimulation of 100 nmol/l Ang II. (B) Differences between genotypes in intracellular calcium changes in response to Ang II in the absence of CNP or cANF4–23. Differences between genotypes in intracellular calcium level changes in response to Ang II (C) and drug inhibitory effect (D) in the presence of CNP or cANF4–23. Mean±SEM, *P <0.05, **P <0.01.

Figure 7.. The BP-elevating allele at NPR3 locus enhances Ang II-induced human VSMCs contraction. ‘E’ indicates the BP-elevating allele, ‘L’ denotes the BP-lowering allele. (A) Representative images of VSMCs of the three different genotypes at baseline (before stimulation) and 30 min after treatment with 100 nmol/l Ang II, respectively. White dash lines show the original size of cells at baseline, and the black ones outline the sizes at 30 min in the merged panel. Scale bars = 50 µm. (B) Differences in 100 nmol/l Ang II stimulated VSMC contraction of different genotypes at 15, 30, 45, 60 min. Mean ± SEM, *P < 0.05.