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

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

Role of nitric oxide and the endothelium.

<p>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, <i>p</i><0.05). <b>B</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. <b>C</b>) 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, <i>p</i><0.05).</p

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

<p>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>B</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, <i>p</i><0.05), #(maximal efficacy of sFlt-1 vs. sFlt-1 + indo, N = 9–10, <i>p</i><0.05), $(EC₅₀ of sFlt-1 vs. sFlt-1 + indo and Cntrl vs. Cntrl + indo, <i>p</i><0.05)</p

Plasma ceramide is increased and associated with proteinuria in women with pre-eclampsia and HELLP syndrome

Objectives: Ceramide is a sphingolipid with anti-angiogenic and pro-apoptotic properties that has shown to be increased in plasma of women with pre-eclampsia. We aimed to compare plasma and placental sphingolipid content among normotensive pregnant women and pre-eclamptic women with and without HELLP syndrome and we aimed to assess whether ceramide is related to hypertension and proteinuria in pre-eclampsia. Study design: Case-control study. Participants were recruited from the Department of Obstetrics at the Academic Medical Center in Amsterdam, The Netherlands. In total 48 pregnant women were included: 24 with pre-eclampsia and 24 normotensive controls. Of the 24 pre-eclamptic women, 11 had HELLP syndrome. Main outcome measures: Plasma and placental ceramide content and correlation with blood pressure and protein excretion in pre-eclampsia. Results: Total plasma, but not placental, ceramide was higher in pre-eclamptic women with HELLP syndrome (11200 95% CI 9531–12870 nmol/ml, n = 11) compared to pre-eclamptic women without HELLP (7413 95% CI 5928–8898 nmol/ml, n = 13, p < 0.001) and normotensive pregnant women (7404 95% CI 6695–8112 nmol/ml, n = 24, p < 0.001). Maternal circulating ceramide levels were strongly associated with proteinuria (r = 0.621, n = 24, p = 0.001) in pre-eclamptic women and inversely correlated with gestational age at delivery (r = 0.771, p < 0.01) in pre-eclamptic women with HELLP syndrome. Plasma ceramide was not correlated with blood pressure. Conclusion: Plasma but not placental ceramide content is increased in women with pre-eclampsia and HELLP syndrome. The strong positive correlation with proteinuria and the inverse correlation with gestational age at delivery indicate that excess plasma ceramide may contribute to the pathophysiology of pre-eclampsia and HELLP

Effect of sFlt-1 on blood pressure.

<p><i>In vivo</i> 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, * <i>p</i><0.05.</p

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

<p>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, <i>p</i><0.05), #(high-responders vs. low-responders, N = 7–5, <i>p</i> = 0.06). <b>B</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, <i>p</i><0.05), $(EC₅₀ of sFlt-1 vs. sFlt-1 + BQ788 and Cntrl vs. Cntrl + BQ788, <i>p</i><0.05)</p

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

<p>The <i>in vivo</i> 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, <i>p</i><0.05 with one-way anova and Dunnets post-hoc test.</p