SFlt-1 elevates blood pressure by augmenting endothelin-1-mediated vasoconstriction in mice

Abstract

Objective: Scavenging of vascular endothelial growth factor (VEGF) elevates blood pressure (BP) in patients receiving anti-angiogenic therapy. Similarly, inhibition of circulation VEGF by its soluble receptor fms-like tyrosine kinase-1 (sFlt-1) underlies BP elevation in pre-eclampsia. Both phenotypes are characterized by augmented production of endothelin-1 (ET-1), suggesting a role for ET-1 in anti-angiogenic hypertension. We aimed to assess the effect of VEGF inhibition on ET-1-induced contractility and downstream ET-1 signaling.

Approach and results: Male C57BL/6N mice were treated with either sFlt-1 or vehicle and BP was assessed via tail-cuff. Mean arterial pressure of sFlt-1-treated mice markedly increased compared to vehicle-treated controls (N = 11-12, p<0.05). After sacrifice, carotid and mesenteric arteries were isolated for isometric tension measurements. ET-1-induced contractions were similar in mesenteric arteries of vehicle and sFlt-1-treated mice, but augmented in carotid segments of sFlt-1-treated mice compared to controls (N = 9-10, p<0.05). The increased contraction in carotid segments could be completely abrogated by the cyclooxygenase (COX) inhibitor indomethacin (N = 9-10, p<0.05), indicating heightened prostaglandin-mediated vasoconstriction. This was associated with a shift towards procontractile ETB signaling in sFlt-1-treated mice, possibly explaining the increased ET-1-induced prostaglandin-mediated vasoconstriction. In line with the ex vivo findings, sFlt-1-induced BP elevation could be prevented in vivo by oral treatment with either a high-dose of the COX inhibitor aspirin (N = 7) or with picotamide (N = 9), a dual thromboxane A2 synthase inhibitor and receptor antagonist.

Conclusions: VEGF inhibition augments the pressor response to ET-1. The cyclooxygenase-thromboxane signaling route downstream of ET-1 might be a possible target to prevent BP elevation during VEGF inhibition.

Figure 1. Effect of sFlt-1 on blood pressure.

In vivo effect of sFlt-1 or vehicle (Cntrl) infusion during two weeks on mean arterial pressure (MAP). Data are presented as mean±SEM, N = 11–12, (ns) not significant, * p<0.05.

Figure 2. Effect of COX inhibition on endothelin-1 concentration-response curves.

A) Concentration-response curve of endothelin-1 in isolated carotid arteries of vehicle-treated (Cntrl) and sFlt-1-treated mice. The inset depicts concentration-response curves of KCl (N = 15) and the α1-adrenergic receptor agonist phenylephrine (N = 8–9). B) Effect of pre-incubation with the non-selective cyclooxygenase-inhibitor indomethacin (indo; 10 μmol/L) on endothelin-1 concentration-response curves. Data are expressed as mean±SEM, *(maximal efficacy of sFlt-1 vs. Cntrl, N = 9–10, p<0.05), #(maximal efficacy of sFlt-1 vs. sFlt-1 + indo, N = 9–10, p<0.05), $(EC₅₀ of sFlt-1 vs. sFlt-1 + indo and Cntrl vs. Cntrl + indo, p<0.05)

Figure 3. Role of nitric oxide and the endothelium.

A) Effect of eNOS inhibition with L-NAME (N = 5) and endothelium (EC) denudation (N = 4) on ET-1 concentration-response curves in carotid segments of vehicle-treated mice (Cntrl). Data are expressed as mean±SEM, *(maximal efficacy of Cntrl vs. Cntrl + L-NAME and Cntrl vs. Cntrl + EC denuded, N = 9–5, p<0.05). B) Effect of L-NAME (N = 4) and EC denudation (N = 3) on ET-1 concentration-response curves in carotid segments of sFlt-1-treated mice. C) Metacholine concentration-response curve generated after pre-constriction with the α1-adrenergic receptor agonist phenylephrine. Data are expressed as mean±SEM,*(maximal relaxation sFlt-1 vs. Cntrl, N = 13–14, p<0.05).

Figure 4. mRNA expression of ET_A and ET_B receptors and effect ET_B receptor blockade.

A) Quantitative Real-Time PCR showing thoracic aorta mRNA expression profiles of Ednra and Ednrb in vehicle (Cntrl) and sFlt-1-treated mice normalized to Hprt mRNA levels. Data expressed as mean±SEM, *(Inset figure shows a two-fold increase in high-responders vs. Cntrl, N =  7–13, p<0.05), #(high-responders vs. low-responders, N = 7–5, p = 0.06). B) Effect of pre-incubation with the ET_B receptor antagonist BQ788 on endothelin-1 concentration-response curves of carotid segments isolated from vehicle (Cntrl, N = 5) and sFlt-1-treated mice (N = 4). *(maximal efficacy of Cntrl vs. Cntrl + BQ788, N = 5–9, p<0.05), $(EC₅₀ of sFlt-1 vs. sFlt-1 + BQ788 and Cntrl vs. Cntrl + BQ788, p<0.05)

Figure 5. Effect of aspirin and picotamide on sFlt-1-induced BP elevation.

The in vivo effect of intervention with aspirin (30 mg/kg/day) or picotamide (5 mg/kg/day) during two weeks on mean arterial pressure (MAP). Data are expressed as mean±SEM, N = 7–12. *(sFlt-1 vs Cntrl, p<0.05 with one-way anova and Dunnets post-hoc test.