Angiogenic growth factors are new and essential players in the sustained relaxin vasodilatory pathway in rodents and humans

Abstract

Relaxin is emerging as an important vasodilator of pregnancy and is being tested for afterload reduction in acute heart failure. However, the mechanisms underlying relaxin-induced vasodilation are incompletely understood. The aims of this study were to establish a new in vitro model for relaxin-induced vasodilation and to use this approach, as well as chronically instrumented, conscious rats, to investigate the role of angiogenic growth factors in the relaxin vasodilatory pathway. Incubation of rat and mouse small renal arteries with recombinant human H2 relaxin for 3 hours in vitro attenuated myogenic constriction, which was blocked by inhibitors of gelatinases, the endothelin B receptor, and NO synthase. These findings corroborate ex vivo observations in arteries isolated from relaxin-infused nonpregnant and midterm pregnant rats, thereby validating the new experimental approach and enabling the study of human arteries. Incubation of small human subcutaneous arteries with relaxin for 3 hours in vitro also attenuated myogenic constriction through the same molecular intermediates. Vascular endothelial growth factor receptor inhibitor SU5416, 3 different vascular endothelial growth factor, and 2 different placental growth factor neutralizing antibodies prevented relaxin from attenuating myogenic constriction in rat and mouse small renal and human subcutaneous arteries. SU5416 administration also prevented relaxin-induced renal vasodilation and hyperfiltration in chronically instrumented, conscious rats. Small renal arteries isolated from these rats demonstrated increased matrix metalloproteinase 2 activity in the relaxin-infused group, which was not prevented by SU5416. We conclude that there is concordance of relaxin vasodilatory mechanisms in rats, mice, and humans, and angiogenic growth factors are novel and essential intermediates.

Figure 1. Timeline for the renal function study in chronically instrumented, conscious rats

The experiment was designed to test the hypothesis that angiogenic growth factor receptor(s) are involved in relaxin-induced increases in effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in conscious female rats. Mean arterial pressure (MAP) was also assessed. Note that osmotic pumps containing either recombinant human relaxin (rhRLX) or dilute sodium acetate vehicle were primed prior to implantation, so that infusion commenced immediately.

Figure 2. The VEGF receptor tyrosine kinase inhibitor SU5416 prevents attenuation of myogenic constriction by relaxin in isolated arteries

(A) Rat small renal and (B) human subcutaneous arteries were pretreated for 30 min with SU5416 (1 μmol/L) or dilute dimethylsulfoxide (DMSO) vehicle and incubated with recombinant human relaxin (rhRLX; 30 ng/ml) for 3 hr. (C) Additional rat small renal arteries were first treated with rhRLX for 3 hr, then SU5416 or DMSO was added to the bath for 30 min in the continuing presence of rhRLX before assessing myogenic constriction (P=NS). *P=0.05, †P<0.005 SU5416 vs DMSO. The initial diameters (μm) were: (A) SU5416 225.1±24.9, DMSO 210.0±14.0; (B) SU5416 281.7±26.3, DMSO 201.7±64.6, and (C) rhRLX/SU5416 245.4±30.5, rhRLX/DMSO 245.5±16.3 (all P=NS by Wilcoxon signed-ranks or Krusal-Wallis test).

Figure 3. VEGF and PGF immunoneutralization prevents attenuation of myogenic constriction by relaxin in isolated arteries

(A) Mouse and (B) rat small renal, and (C) human subcutaneous arteries were pretreated for 30 minutes with specific VEGF-neutralizing or goat IgG isotype control antibodies (1 – 3 μg/ml instilled internally) and incubated with recombinant human relaxin (rhRLX; 30 ng/ml) for 3 hr. (D) Mouse and (E) rat small renal, and (F) human subcutaneous arteries were pretreated for 30 min with specific PGF-neutralizing or rat IgG_2A & mouse IgG₁ isotype control antibodies (0.1 – 10 μg/ml; instilled internally) and incubated with rhRLX (30 ng/ml) for 3 hr. For additional antibody details and concentrations, see Supplemental Table 2. *P<0.05 †P<0.01 ‡P<0.001 isotype control vs VEGF or PGF antibody (Ab). The initial diameters (μm) were: (A) VEGF Ab 178.1±16.6, Goat IgG 172.3±20.6; (B) VEGF Ab 245.2±15.1, Goat IgG 247.9±14.1; (C) VEGF Ab 178.1±36.4, Goat IgG 276.5±78.5; (D) PGF Ab 162.4±21.2, Rat IgG_2A 143.6±8.4; (E) PGF Ab 265.7±44.8 Rat IgG_2A 264.3±18.3 (all P=NS by Krusal-Wallis test) and (F) PGF Ab 492.0±73.0 (n=2), Mouse IgG₁ 534 (n=1).

Figure 4. Effect of angiogenic growth factor inhibition on endothelial function

After assessment of myogenic constriction in isolated rat small renal arteries pre-incubated with (A) SU5416 or dilute DMSO vehicle, (B) PGF or (C) VEGF neutralizing or control antibodies, and then incubated with rhRLX (30 ng/ml) for 3 hr, internal pressure was returned to 60 mmHg and arteries were re-equilibrated in fresh buffer for 15 min. Arteries were then constricted to 50% of their initial diameter with phenylephrine, and methacholine was added at either a maximal dose (1×10⁻⁶M; A) or in dose-response fashion (B & C). Responses to methacholine are expressed as % relaxation from the phenylephrine-constricted value. Endothelial function was either not compromised in the case of SU5416, slightly enhanced by PGF blockade (P=0.0127), or slightly decreased by VEGF immunoneutralization (P<0.001). P values were obtained by t-test (A) or two way ANOVA (P values in brackets) with LSD post-hoc tests (B & C). *P < 0.05 vs control antibody.

Figure 5. VEGF receptor tyrosine kinase inhibition with SU5416 prevents relaxin-induced renal vasodilation and hyperfiltration

Treatment with SU5416 (20 mg/kg/d s.c.) blocked the increases in (A) glomerular filtration rate and (B) effective renal plasma flow, and decreases in (C) effective renal vascular resistance induced by recombinant human relaxin (rhRLX; 4 μg/h) infusion for 4–6 hr and 3–5 days without affecting (D) mean arterial pressure (left panels). Control animals received vehicle (20 mM sodium acetate, pH 5) instead of rhRLX (right panels). *P<0.05 vs baseline, †P<0.05 vs SU5416 group by two way ANOVA with LSD post-hoc tests.

Figure 6. MMP-2 activity in small renal arteries assessed by gelatin zymography

After the last measurement of MAP and renal function (see Figure 1), rats were euthanized and small renal arteries harvested. Upper panels in (A) and (B) depict representative zymograms. The densitometric ratios of rhRLX:vehicle and SU5416:DMSO are shown in the lower panels (n = 7–9 rats in each of the 4 treatment groups). (A) Recombinant human relaxin (RLX; 4 μg/hr) administered in vivo increased pro + active MMP-2 activity compared to sodium acetate vehicle (VEH) in the small renal arteries of rats receiving daily injections of DMSO. *P=0.028 vs 1.0 by one-sample t-test. (B) Pro MMP-2 activity was not consistently different in the small renal arteries of rats treated with rhRLX + SU5416 (SU) compared to rhRLX + DMSO. (Note that active MMP-2 band intensity was too low to be quantified for this comparison.) STD: MMP-2 standard.

Figure 7. Working model for the sustained vasodilatory effect of relaxin

The precise localization of VEGF and PGF in the relaxin vasodilatory pathway is currently unknown (?); relaxin may increase expression of angiogenic growth factor(s) in the arterial wall and/or release them from the extracellular matrix via MMP-9 or -2. Inhibitors of pregnancy- and/or relaxin-induced vasodilation are shown in the boxes. ET, endothelin; MMP, matrix metalloproteinase; ECM, extracellular matrix; RBF, renal blood flow; GFR, glomerular filtration rate; RXFP, relaxin/insulin-like family peptide receptors; SU5416, vascular endothelial growth factor receptor tyrosine kinase inhibitor; GM6001, a general MMP inhibitor; cyclic CTT, a specific peptide inhibitor of MMP-2; TIMP-2, tissue inhibitor of matrix metalloproteinase; RES-701-1, a specific ET_B receptor antagonist; SB209670, a mixed ET_A and ET_B receptor antagonist; L-NAME, nitro-L-arginine methyl ester; L-NMMA, N^G-monomethyl-L-arginine. Note that RXFP2 knockout (in mice), STT (control peptide for cyclic CTT), heat inactivated TIMP-2, BQ-123 (a specific ET_A receptor antagonist), phosphoramidon (an inhibitor of the classical endothelin converting enzyme), D-NAME and isotype-matched IgGs (controls for neutralizing antibodies) did not affect the sustained vasodilatory responses to relaxin. See text for details and supporting references.