Restoring placental growth factor-soluble fms-like tyrosine kinase-1 balance reverses vascular hyper-reactivity and hypertension in pregnancy

Abstract

Preeclampsia (PE) is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism. An imbalance between antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and angiogenic placental growth factor (PlGF) has been observed in PE, but the vascular targets and signaling pathways involved are unclear. We assessed the extent of sFlt-1/PlGF imbalance and vascular dysfunction in a rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and tested whether inducing a comparable sFlt-1/PlGF imbalance by infusing sFlt-1 (10 μg·kg(-1)·day(-1)) in day 14 pregnant (Preg) rats cause similar increases in blood pressure (BP) and vascular reactivity. Using these guiding measurements, we then tested whether restoring sFlt-1/PlGF balance by infusing PIGF (20 μg·kg(-1)·day(-1)) in RUPP rats would improve BP and vascular function. On gestational day 19, BP was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP rats. Plasma sFlt-1/PlGF ratio was increased in Preg+sFlt-1, and RUPP and was reduced in RUPP+PlGF rats. In isolated endothelium-intact aorta, carotid, mesenteric, and renal artery, phenylephrine (Phe)- and high KCl-induced contraction was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP. The differences in vascular reactivity to Phe and KCl between groups were less apparent in vessels treated with the nitric oxide synthase (NOS) inhibitor l-NAME or guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or endothelium-denuded, suggesting changes in endothelial NO-cGMP pathway. In Phe precontracted vessels, ACh-induced relaxation was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP, and was blocked by N(ω)-nitro-l-arginine methyl ester (l-NAME) or ODQ treatment or endothelium removal. Western blots revealed that aortic total endothelial NOS (eNOS) and activated phosphorylated-eNOS were in Preg+sFlt-1 and RUPP < Preg and in RUPP+PlGF > RUPP. ACh-induced vascular nitrate/nitrite production was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP. Vascular relaxation to the exogenous NO donor sodium nitroprusside was not different among groups. Thus, a tilt in the angiogenic balance toward anti-angiogenic sFlt-1 is associated with decreased vascular relaxation and increased vasoconstriction and BP. Restoring the angiogenic/antiangiogenic balance using PlGF enhances endothelial NO-cGMP vascular relaxation and decreases vasoconstriction and BP in HTN-Preg rats and could offer a new approach in the management of PE.

Keywords: angiogenesis; calcium; endothelium; hypertension; nitric oxide; preeclampsia; vascular smooth muscle.

Fig. 1.

Maternal and fetal parameters in pregnant (Preg), Preg+soluble fms-like tyrosine kinase-1 (sFlt-1), reduced uteroplacental perfusion pressure (RUPP), and RUPP+placental growth factor (PlGF) rats. On day 19 of pregnancy, maternal plasma sFlt-1 (A), PlGF (B), sFlt-1/PIGF ratio (C), and blood pressure (BP) (D), as well as litter size (E), and average pup weight (F) were compared in Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats. Data are expressed as means ± SE; n = 6 or 7. *P < 0.05, Preg+sFlt-1 or RUPP vs. Preg. #P < 0.05, RUPP+PlGF vs. RUPP.

Fig. 2.

KCl-induced contraction in blood vessels of Preg, Preg+sFlt-1, RUPP and RUPP+PlGF rats. KCl (96 mM)-induced contraction was compared in endothelium-intact (A–D) and denuded (–Endo) (E–H) aorta, carotid, mesenteric, and renal artery of Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats, Data represent means ± SE; n = 5–9. *P < 0.05, Preg+sFlt-1 or RUPP vs. Preg. #P < 0.05, RUPP+PlGF vs. RUPP.

Fig. 3.

Phe-induced contraction in blood vessels of Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats. Endothelium-intact aortic, carotid, mesenteric and renal artery rings from Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats were stimulated with increasing concentrations of Phe, the contractile response was measured and presented in grams (g) (A–D) as % of maximum Phe contraction (E–H), or as % of KCl contraction (I–L). Data represent means ± SE; n = 5–9. *P < 0.05, Preg+sFlt-1 or RUPP vs. Preg. #P < 0.05, RUPP+PlGF vs. RUPP.

Fig. 4.

Effect of endothelium removal or blockade of NO-cGMP pathway on Phe-induced contraction in grams (g). Isolated aortic, carotid, mesenteric and renal artery rings from Preg (A–D), Preg+sFlt-1 (E–H), RUPP (I–L), and RUPP+PlGF (M–P) rats were either kept endothelium-intact (○), pretreated with the NOS inhibitor l-NAME (3×10⁻¹ M) (●) or the guanylate cyclase inhibitor ODQ (10⁻⁵ M) (△) for 15 min, or endothelium-denuded (▲). The vessels were stimulated with increasing concentrations of Phe, and the contractile response was presented in grams (g). Data represent means ± SE; n = 5–9. *P < 0.05, N_ω-nitro-l-arginine methyl ester (l-NAME) treated vs. nontreated vessels. #P < 0.05, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-treated vs. nontreated vessels. †Endothelium-denuded (−Endo) vs. intact (+Endo) vessels.

Fig. 5.

Effect of endothelium removal or blockade of nitric oxide (NO)-cGMP pathway on sensitivity to Phe. Aortic, carotid, mesenteric, and renal artery rings from Preg (A–D), Preg+sFlt-1 (E–H), RUPP (I–L), and RUPP+PlGF (M–P) rats were either kept endothelium-intact (○), pretreated with the NOS inhibitor l-NAME (3×10⁻⁴ M) (●) or guanylate cyclase inhibitor ODQ (10⁻⁵ M) (△), or endothelium-denuded (▲). The vessels were stimulated with increasing concentrations of Phe, and the contractile response was presented as % of maximum Phe contraction. Data represent means ± SE; n = 5–9. *P < 0.05, l-NAME treated vs. nontreated vessels. #P < 0.05, ODQ-treated vs. nontreated vessels.

Fig. 6.

Effect of endothelium removal or blockade of NO-cGMP on Phe-induced contraction relative to control KCl contraction. Aortic, carotid, mesenteric, and renal artery rings from Preg (A–D), Preg+sFlt-1 (E–H), RUPP (I–L), and RUPP+PlGF rats were either kept intact (○), pretreated with the NOS inhibitor l-NAME (3×10⁻⁴ M) (●) or guanylate cyclase inhibitor ODQ (10⁻⁵ M) (△), or endothelium-denuded (▲). After measuring contraction to 96 mM KCl, the vessels were stimulated with increasing concentrations of Phe, and the contractile response was presented as % of KCl contraction. Data represent means ± SE; n = 5–9. *P < 0.05, l-NAME treated vs. nontreated vessels. #P < 0.05, ODQ-treated vs. nontreated vessels. †Endothelium-denuded (−Endo) vs. intact (+Endo) vessels.

Fig. 7.

ACh-induced relaxation in blood vessels of Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats. Endothelium-intact aortic (A), carotid (B), mesenteric (C), and renal artery rings (D) from Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats were precontracted with submaximal concentration of Phe, and then stimulated with ACh (10⁻⁹-10⁻⁵ M), and the % relaxation of Phe contraction was measured. Data represent means ± SE; n = 6–9. *P < 0.05, Preg+sFlt-1 or RUPP vs. Preg. #P < 0.05, RUPP+PlGF vs. RUPP.

Fig. 8.

Role of endothelial NO-cGMP pathway in ACh-induced vascular relaxation. Isolated aortic, carotid, mesenteric, and renal artery rings from Preg (A–D), Preg+sFlt-1 (E–H), RUPP (I–L), and RUPP+PlGF rats (M–P) were either kept endothelium-intact (○), pretreated with the NOS inhibitor l-NAME (3×10⁻⁴ M) (●), or the guanylate cyclase inhibitor ODQ (10⁻⁵ M) (△) for 10 min, or endothelium-denuded (▲). The vessels were precontracted with submaximal concentration of Phe and then stimulated with ACh (10⁻⁹-10⁻⁵ M), and the % relaxation of Phe contraction was measured. Data represent means ± SE; n = 6–9. *P < 0.05, l-NAME treated, ODQ-treated or endothelium-denuded (-Endo) vs. control nontreated endothelium-intact vessels.

Fig. 9.

Protein amount of eNOS and NO production in blood vessels of Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats. Aortic tissue homogenate from Preg, Preg+sFlt-1, RUPP and RUPP+PlGF rats were prepared for Western blots using endothelial nitric oxide synthase (eNOS) and p-eNOS antibodies (1:800), and both total eNOS (A) and activated p-eNOS (B) were measured. In parallel experiments, ACh induced nitrate/nitrite (NOx) production was measured in the aorta (C) and mesenteric artery (D) of different animal groups. Data represent means ± SE; n = 4–7. *P < 0.05, Preg+sFlt-1 or RUPP vs. Preg. #P < 0.05, RUPP+PlGF vs. RUPP.

Fig. 10.

SNP-induced relaxation in blood vessels of Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats. Endothelium-denuded aortic (A), carotid, (B) mesenteric (C), and renal artery rings (D) from Preg, Preg+sFlt-1, RUPP, and RUPP+PlGF rats were precontracted with submaximal concentration of Phe, then stimulated with SNP (10⁻⁹-10⁻⁵ M) and the % relaxation of Phe contraction was measured. Data are expressed as means ± SE; n = 6–9.