A rare genetic variant of BPIFB4 predisposes to high blood pressure via impairment of nitric oxide signaling

Abstract

BPIFB4 is associated with exceptional longevity: four single-nucleotide polymorphisms distinguish the wild-type form from a longevity-associated variant conferring positive effects on blood pressure. The effect of a rare variant (RV; allele frequency, 4%) on blood pressure is unknown. Here, we show that overexpression of RV-BPIFB4 in ex-vivo mouse vessels impairs phosphorylation of endothelial nitric oxide synthase (eNOS), blunting acetylcholine-evoked vasorelaxation; in vivo, virally mediated overexpression of RV-BPIFB4 increases blood pressure, an action absent in eNOS-deficient mice. In humans, we found RV carriers to have increased diastolic blood pressure, a finding that was more marked in subjects on anti-hypertensive medication; moreover, recombinant RV-BPIFB4 protein impaired eNOS function in ex-vivo human vessels. Thus, RV-BPIFB4 acts directly on blood pressure homeostasis and may represent a novel biomarker of vascular dysfunction and hypertension.

Figure 1

Response of ex vivo mouse mesenteric arteries to transfection with RV-BPIFB4. (A,B) Graphs of vascular response to increasing doses of acetylcholine (ACh) or nitroglycerine in mouse mesenteric arteries before and after transfection with plasmids encoding WT-BPIFB4. (C,D) Graphs of vascular response to increasing doses of acetylcholine (ACh) or nitroglycerine in mouse mesenteric arteries before and after transfection with plasmids encoding RV-BPIFB4 (n = 7 for each experiment). *p < 0.05; **p < 0.01;***p < 0.001 (two-way ANOVA followed by Bonferroni post hoc test). (E) left, Representative immunoblot of mesenteric artery lysate collected after vascular reactivity studies; right, Optical density measurements of immunoblottings. Columns are the mean ± SEM of 3 independent experiments. *p < 0.05 (one-way ANOVA analysis followed by Bonferroni post hoc test). Images were cropped using Adobe Photoshop, full-length blots are presented in Supplementary Fig. 2.

Figure 2

In vivo infection of mice with RV-BPIFB4. (A) Graph of systolic blood pressure (SBP) in C57BL/6 mice infected with AAV vectors encoding WT-BPIFB4, RV-BPIFB4, or green fluorescent protein (GFP). The arrow indicates injection of AAV in the femoral artery (day 0). Data are mean ± SEM (n = 6/group). *p < 0.05; **p < 0.01 vs. AVV-WT-BPIFB4; ^##p < 0.01 vs. AVV-GFP (two-way ANOVA followed by Bonferroni post hoc test). (B) representative immunoblottings (left) and optical density measurements (right) of mesenteric arteries harvested from infected mice. Columns are the mean ± SD of 3 independent experiments.*p < 0.05 (one-way ANOVA analysis followed by Bonferroni post hoc test). Images were cropped using Adobe Photoshop, full-length blots are presented in Supplementary Fig. 3. (C,D) ex-vivo response of mesenteric arteries removed from mice after infection with AAV-WT-BPIFB4, AAV-RV-BPIFB4, or AAV-GFP to increasing doses of acetylcholine (ACh) or nitroglycerine. Two-way ANOVA, in combination with Bonferroni post-tests. (E) Graph of systolic blood pressure (SBP) in eNOS knock-out mice (eNOS^−/−) and wild-type mice (eNOS^+/+) infected with AAV vectors encoding RV-BPIFB4 or green fluorescent protein (GFP). The arrow indicates injection of AAV in the femoral artery (day 0). Data are mean ± SEM (n = 5/group). *p < 0.05; **p < 0.01 vs. AVV-GFP + eNOS^+/+ (two-way ANOVA followed by Bonferroni post hoc test). (F) Graph of ex-vivo vascular response in mouse mesenteric arteries removed from wild-type C57BL/6 (eNOS^+/+) and eNOS knock-out (eNOS^−/−) mice infected with AAV-RV-BPIFB4 or AAV-GFP, to increasing doses of acetylcholine (ACh). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01 vs. all (two-way ANOVA followed by Bonferroni post hoc test).

Figure 3

Reaction of ex vivo human arteries to exposure to recombinant WT- and RV-BPIFB4 proteins. (A,B) Dose-response of human superior thyroid arteries (STA) incubated for 1 hour with 18ng/mL of recombinant WT-BPIFB4 (A) or RV-BPIFB4 (B) protein to increasing doses of acetylcholine (Ach). Values are means ± SEM (n = 4 experiments).**p < 0.01 (two-way ANOVA followed by Bonferroni post hoc test). (C) left, Representative immunoblot of human STA after vascular reactivity studies; right, Columns are the mean ± SEM of 3 independent experiments. *p < 0.05 (Student’s t-test). Images were cropped using Adobe Photoshop, full-length blots are presented in Supplementary Fig. 4.