Central administration of TRV027 improves baroreflex sensitivity and vascular reactivity in spontaneously hypertensive rats

Abstract

TRV027 is a biased agonist for the Angiotensin (Ang)-II type 1 receptor (AT₁R), able to recruit β-arrestin 2 independently of G-proteins activation. β-arrestin activation in the central nervous system (CNS) was suggested to oppose the effects of Ang-II. The present study evaluates the effect of central infusion of TRV027 on arterial pressure (AP), autonomic function, baroreflex sensitivity (BRS), and peripheral vascular reactivity. Spontaneously hypertensive (SH) and Wistar Kyoto (WKY) rats were treated with TRV027 for 14 days (20 ng/h) delivered to the lateral ventricle via osmotic minipumps. Mechanistic studies were performed in HEK293T cells co-transfected with AT₁R and Ang converting enzyme type 2 (ACE2) treated with TRV027 (100 nM) or Ang-II (100 nM). TRV027 infusion in SH rats (SHR) reduced AP (~20 mmHg, P<0.05), sympathetic vasomotor activity (ΔMAP = -47.2 ± 2.8 compared with -64 ± 5.1 mmHg, P<0.05) and low-frequency (LF) oscillations of AP (1.7 ± 0.2 compared with 5.8 ± 0.4 mmHg, P<0.05) compared with the SHR control group. TRV027 also increased vagal tone, improved BRS, reduced the reactivity of mesenteric arteries to Ang-II and increased vascular sensitivity to phenylephrine (Phe), acetylcholine, (ACh), and sodium nitroprusside (SNP). In vitro, TRV027 prevented the Ang-II-induced up-regulation of ADAM17 and in contrast with Ang-II, had no effects on ACE2 activity and expression levels. Furthermore, TRV027 induced lesser interactions between AT₁R and ACE2 compared with Ang-II. Together, these data suggest that due to its biased activity for the β-arrestin pathway, TRV027 has beneficial effects within the CNS on hypertension, autonomic and vascular function, possibly through preserving ACE2 compensatory activity in neurones.

Keywords: ACE2; ADAM17; Biased agonist; hypertension; renin-angiotensin system.

Figure 1.. Effect of ICV administration of TRV027 in rats

(A) Typical PAP, MAP, and HR traces from conscious and freely moving rats treated or not with TRV027. TRV027 administration induces a reduction in MAP (B) and SBP (C) in SHR without affecting DBP (D) and HR (E). *P<0.05 compared with WKY control, and #P<0.05 compared with SHR. All data are mean ± S.E.M. WKY: n=9; WKY + TRV: n=7; SHR and SHR + TRV: n=6/ group.

Figure 2.. Evaluation of autonomic function after treatment with TRV027

SHR treated with TRV027 had improved cardiac vagal tone (A) and reduced vascular sympathetic tone (B) although cardiac sympathetic drive was not altered (C). Representative spectra of SBP (D), average magnitudes of LF (E) and HF (F) components of SBP from WKY and SHR treated or not with TRV027. *P<0.05 compared with WKY control, and #P<0.05 compared with SHR. All data are mean ± S.E.M. n=5 in all groups.

Figure 3.. Evaluation of BRS after treatment with TRV027

Both baroreflex gain using a pharmacological method (A) and the spontaneous baroreflex (B) were improved after 14 days of treatment with TRV027 in hypertensive rats. *P<0.05 compared with WKY control, and #P<0.05 compared with SHR. All data are mean ± S.E.M. SHR and WKY: n=6; SHR + TRV and WKY + TRV: n=7; spontaneous baroreflex: n=5 in all groups.

Figure 4.. TRV027 decreases vascular reactivity to Ang-II in mesenteric arteries from SHR

Mean of group data showing the increase in tension values (g) after Ang-II in SHR and a reduction following TRV027 treatment (A). Percentage of contraction for Ang-II (B); time course response to Ang-II (C), and relative contraction/time to Ang-II (D). *P<0.05 compared with WKY control, and #P<0.05 compared with SHR. All data are mean ± S.E.M.; n=7 in all groups.

Figure 5.. Impact of TRV027 treatment on vascular reactivity in SHR

TRV027 treatment increased vascular sensitivity to Phe (A), ACh (B), and SNP (C) in mesenteric arteries from SHRs. All data are mean ± S.E.M.; n=8 in all groups.

Figure 6.. Impact of TRV027 treatment on ACE2 activity and expression, ADAM17 activity and ACE2/AT1R interactions

ACE2 enzymatic activity in ACE2 and AT₁R co-transfected HEK293T cells (A). The activity was tested 72 h after transfection, the cells being serum starved 24 h before experiments. Treatments with Ang-II and TRV027 (100 nM each) lasted for 4 h. *P<0.05 compared with control. n=9 from three independent transfections. (B) Top: ACE2 total cellular levels in HEK293T cells co-transfected with AT₁R and ACE2 for 72 h determined by Western blot. After serum starvation for 24 h, the cells were treated with Ang-II or TRV027 (100 nM each) for 18 h. Bottom: statistics of two independent experiments in two separate transfections. (C) Neuro2A cells were treated with Ang-II (300 nM) and TRV027 (500 nM) for 18 h, then processed for ADAM17 enzymatic assay. In the co-treatment group, Ang-II and TRV027 were added simultaneously to the serum-free medium. (D) ACE2/AT₁R interactions determined by nanoBRET. HEK293T cells were co-transfected with ACE2-NLuc and AT₁R-HaloTag and plated in black 96-well plates at a density of 25 × 10⁴ cells/well for 48 h. One day before measurement, the cells were incubated in DMEM without Phenol Red or FBS. HaloTag NanoBRET 618 Ligand (100 nM final concentration) or DMSO (negative control) were added 16 h before final determination. The cells were treated with 100 nM Ang-II or TRV027 for 4 h. Nano-Glo Substrate was added for 30 min and donor emission (460 nm) and acceptor emission (618 nm) were measured in a Biotek3 spectrophotometer. The final values were calculated as ratio of 618 nm/460 nm after subtraction of the negative control values (DMSO). n=12 from three independent transfections.