Interference With Endothelial PPAR (Peroxisome Proliferator-Activated Receptor)-γ Causes Accelerated Cerebral Vascular Dysfunction in Response to Endogenous Renin-Angiotensin System Activation

Abstract

Low-salt diet is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes mellitus, or other cardiovascular abnormalities because of endogenous renin-angiotensin system activation. PPAR (peroxisome proliferator-activated receptor)-γ is a transcription factor which promotes an antioxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPAR-γ selectively in the endothelium (E-V290M) to test the hypothesis that endothelial PPAR-γ plays a protective role in response to low salt-mediated renin-angiotensin system activation. Plasma renin and Ang II (angiotensin II) were significantly and equally increased in all mice fed low salt for 6 weeks. Vasorelaxation to acetylcholine was not affected in basilar artery from E-V290M at baseline but was significantly and selectively impaired in E-V290M after low salt. Unlike basilar artery, low salt was not sufficient to induce vascular dysfunction in carotid artery or aorta. Endothelial dysfunction in the basilar artery from E-V290M mice fed low salt was attenuated by scavengers of superoxide, inhibitors of NADPH oxidase, or blockade of the Ang II AT1 (angiotensin type-1) receptor. Simultaneous AT1 and AT2 receptor blockade revealed that the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation. We conclude that interference with PPAR-γ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to low salt-mediated activation of the endogenous renin-angiotensin system, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPAR-γ activity and renin-angiotensin system activation.

Keywords: endothelial dysfunction; hypertension; mice; renin-angiotensin system; risk factors; sodium restriction.

Figure 1.. RAS Activation

A) Circulating levels of renin measured by ELISA in NT and E-V290M mice (NT chow, n=7; E-V290M chow, n=9; NT LSD, n=8; E-V290M LSD, n=8). B) Plasma levels of angiotensin peptides measured by ELISA in NT and E-V290M mice (NT chow, n=9; E-V290M chow, n=9; NT LSD, n=8; E-V290M LSD, n=9). C) Systolic blood pressure measured using tail-cuff plethysmography in NT and E-V290M mice (n=6 per group). Systolic blood pressure was measured by tail cuff for 6 weeks following 3 weeks of training. Blood pressure was recorded 5 days per week for 6 weeks during the LSD. Data in all panels are presented as mean±SEM with individual data points shown and analyzed by two-way ANOVA. *P<0.05 vs. genotype-matched NT.

Figure 2.. Endothelial Function in Carotid Artery

Dose-dependent relaxation was measured in the carotid arteries from NT and E-V290M mice fed chow or LSD. Cumulative concentration response curves for A) ACh and B) maximum relaxation to ACh (n=5 per group). Cumulative concentration response curves for C) SNP and D) maximum relaxation to SNP (n=5 per group). Data in all panels are presented as mean±SEM analyzed by two-way ANOVA with repeated measures.

Figure 3.. Vascular Function in Basilar Artery

Dose-dependent relaxation was measured in the basilar arteries from NT and E-V290M mice fed chow or LSD. Arteries were pressurized at 60 mmHg and pre-constricted with thromboxane A2 mimetic (U46619). Cumulative concentration response curves for A) ACh and B) maximum relaxation to ACh (NT chow, n=5; E-V290M chow, n=5; NT LSD, n=6; E-V290M LSD, n=8). Cumulative concentration response curves for C) SNP and D) maximum relaxation to SNP (NT chow, n=5; E-V290M chow, n=5; NT LSD, n=6; E-V290M LSD, n=6). Data in panels A-D are presented as mean±SEM analyzed by two-way ANOVA with repeated measures. *P<0.05 E-V290M LSD vs. all other groups. Vasoconstrictive responses to E) Potassium chloride (n=5 per group) and F) Angiotensin II (n=5 per group) in the basilar artery are shown. Data in panels E-F are presented as mean±SEM analyzed by two-way ANOVA. P>0.05.

Figure 4.. Endothelial Function in Basilar Artery: Role of Oxidative Stress.

A) Nitrotyrosine levels measured by ELISA in the pooled cerebral vasculature of chow- and LSD-fed NT and E-V290M mice (n=8). Cumulative concentration response curves for B) ACh in basilar arteries from LSD-fed- NT (n=5) and E-V290M mice (n=7) with and without tempol (1mmol/L, 30 mins); C) ACh in basilar arteries from NT (n=5) and E-V290M mice (n=6) with and without apocynin (100 µmol/L, 30mins); D) ACh in basilar arteries from NT (n=5) and E-V290M mice (n=5) with and without VAS2870 (100 µmol/L, 30mins). Data in all panels are presented as mean±SEM analyzed by two-way ANOVA with repeated measures. *P<0.05 E-V290M LSD vs. all other groups. E) Western blot detecting phosphorylated P47phox (phos-p47phox) and total P47phox in pooled cerebral arteries (NT chow, n=5; E-V290M chow, n=5; NT LSD, n=6; E-V290M LSD, n=5). Quantification of the western blot data is also shown. Data were normalized to the average control value set to 1.0. Data in panel E are mean±SEM analyzed by two-way ANOVA. *P<0.05 E-V290M LSD vs. all other groups.

Figure 5.. Gene expression in cerebral vessels

mRNA expression levels of the indicated genes in pooled cerebral vessels from NT and E-V290M mice fed a LSD or standard chow (n=7 per group). Data in all panels are presented as mean±SEM analyzed by two-way ANOVA. *P<0.05 vs NT LSD and ^$P<0.05 vs. E-V290M chow.

Figure 6.. Endothelial Function in Basilar Artery: Role of AT1 and AT2 Receptors.

A) Circulating levels of renin measured by ELISA in LSD-fed NT and E-V290M mice in response to Losartan administration (NT LSD, n=6; E-V290M LSD, n=6, NT LSD+Los, n=5; E-V290M LSD+Los, n=6), *P<0.05 LSD vs. LSD + Los. B) Systolic blood pressure measured using tail-cuff plethysmography in LSD-fed NT and E-V290M mice in response to Losartan administration (NT LSD, n=5; E-V290M LSD, n=6, NT LSD+Los, n=5; E-V290M LSD+Los, n=6), *P<0.05 LSD vs. LSD + Los. C) Cumulative concentration response curves for ACh and D) maximum relaxation to ACh in basilar arteries from LSD-fed- NT (n=5) and E-V290M mice (n=5) ± losartan. E) mRNA expression levels of the AT1R gene in pooled cerebral vessels from NT and E-V290M mice fed a LSD or standard chow (n=6 per group). F) Cumulative concentration response curves for ACh in basilar arteries from LSD-fed- NT (n=4) and E-V290M mice (n=4) + losartan ± PD123319. Data in all panels are presented as mean±SEM analyzed by two-way ANOVA (A, E) or two-way ANOVA with repeated measures (B, C, D, F). *P<0.05 E-V290M LSD vs. all other groups.