An interaction of renin-angiotensin and kallikrein-kinin systems contributes to vascular hypertrophy in angiotensin II-induced hypertension: in vivo and in vitro studies

Abstract

The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)-induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg(9)-Leu(8)-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184 ± 5.9 vs 115 ± 2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8 ± 2.7 vs 6.0 ± 1.8] and ERK1/2 phosphorylation (% of control: 218.3 ± 29.4 vs 100 ± 0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17 ± 3.1) and ERK1/2 phosphorylation (137 ± 20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings highlight an important cross-talk between the DABK and ANG II in the vascular system and contribute to a better understanding of the mechanisms involved in vascular remodeling in hypertension.

Figure 1. B1 and AT1 receptors expression in aorta and aortic smooth muscle cells (VSMC) primary culture.

Bar graph shows B1 receptor (B1R) [A] and AT1 receptor [B] mRNA levels in aortas from control, ANG II (400 ng/Kg/min), ANG II (400 ng/Kg/min) + DAL (350 ng/Kg/min) and ANG II (400 ng/Kg/min) + LOS (10 mg/Kg/day) rats. The receptors mRNA expression was calculated from the cycle threshold (Ct) value using the Δ²Ct method for quantification. Values were normalized against β-actin mRNA. Data represented mean±SEM; n = 5–7 for each group; *P<0.05 vs control. [C] Bar graph shows the temporal effects of ANG II (100 nM, 0–24 h) on protein B1R expression in aortic VSMC. [D] Bar graph shows B1R expression in aortic VSMC after stimulation with ANG II (100 nM, 2 h), in the presence or not of AT1 antagonist, losartan (LOS – 10 µM). B1R expression was normalized against the housekeeping protein β-actin. Data are presented as mean±SEM of 4 experiments. *P<0.05 vs. control.

Figure 2. B1 receptor antagonism prevented angiotensin II (ANG II) effect in aorta.

[A] Fluorescence microscopy of aortic transverse sections after incubation with DHE. [B] Ratios of 2-hydroxyethidium/dihydroethidine (EOH/DHE) and ethidium/dihydroethidine (E/DHE) obtained by HPLC analysis from aortic segments. Data are expressed as mean ± SEM of 5 rats for each group. *P<0.05 vs control and **P<0.05 vs. ANG II [C] Bar graph shows the ratio of phosphorylated/total ERK1/2 that was used as an indicator of ERK1/2 activity. Data are expressed as mean ± SEM of 6 rats for each group. *P<0.05 vs control and **P<0.05 vs. ANG II.

Figure 3. Effect of angiotensin II (ANG II) and des-Arg⁹-bradykinin (DABK) on superoxide anion generation in VSMC.

[A] Bar graph demonstrates the effects of ANG II (0.1 nM), DABK (0.1 nM) and ANG II-DABK co-stimulation for 5 min, in the presence of either vehicle, AT1 antagonist losartan (LOS – 10 µM), B1 receptor antagonist des-Arg⁹-Leu⁸-bradykinin (DAL – 10 µM) or superoxide anion scavenger (TIRON - 1 mM) on superoxide anion generation. Results were expressed as mean±SEM of 5 experiments. *P<0.05 vs vehicle. [B] Bar graph demonstrates the effects of ANG II (100 nM) stimulation for 5 min, in the presence of vehicle, AT1 antagonist, losartan (LOS – 10 µM), or B1 receptor antagonist, des-Arg⁹-Leu⁸-bradykinin (DAL – 10 µM). Results are represented as mean ± SEM of 5 experiments. *P<0.05 vs vehicle.

Figure 4. Effects of des-Arg⁹-bradykinin (DABK) and angiotensin II (ANG II) on ERK1/2 activation.

[A] Aortic VSMC from Wistar rats treated with ANG II and/or DABK at low concentration (0.1 nM) in the presence of vehicle, losartan (LOS – 10 µM), des-arg9-leu8-bradykinin (DAL – 10 µM) and [B] TIRON (1 mM). Bar graph shows the ratio of phosphorylated/total ERK1/2 that was used as an indicator of ERK1/2 activity. Results are represented as mean±SEM of 5 experiments. *P<0.05 vs Vehicle. [C] Aortic VSMC from Wistar rats were treated with ANG II and/or DABK at high concentration (100 nM). Bar graph showed the ratio of phosphorylated/total ERK1/2 that was used as indicator of ERK1/2 activity. Results are expressed as mean ± SEM of 5 experiments. *P<0.05 vs vehicle.

Figure 5. Effects of des-Arg⁹-bradykinin (DABK) and angiotensin II (ANG II) vascular smooth muscle cells (VSMC) growth.

[A] Aortic VSMC from Wistar rats treated with ANG II and/or DABK at low concentration (0.1 nM) in the presence or not of losartan (LOS – 10 µM), des-arg9-leu8-bradykinin (DAL – 10 µM) and TIRON (1 mM). Bar graph shows PCNA expression normalized against the housekeeping protein β-actin. Results are represented as mean±SEM of 4 experiments. *P<0.05 vs Vehicle. [B] Aortic VSMC from Wistar rats with ANG II at high concentration (100 nM) in the presence or not of losartan (LOS – 10 µM), des-arg9-leu8-bradykinin (DAL – 10 µM). Bar graph shows PCNA expression normalized against the housekeeping protein β-actin. Results are expressed as mean±SEM of 5 experiments. *P<0.05 vs vehicle. [C] [H3] leucine incorporation in VSMC treated with ANG II and/or DABK at low concentration (0.1 nM) in the presence or not of losartan (LOS – 10 µM) and des-arg9-leu8-bradykinin (DAL – 10 µM) and VSMC treated with ANG II at high concentration (100 nM). Results are expressed as mean±SEM of 3 experiments and *P<0.05 vs vehicle.