Calcitonin gene-related peptide inhibits angiotensin II-induced NADPH oxidase-dependent ROS via the Src/STAT3 signalling pathway

Abstract

We had previously demonstrated that the calcitonin gene-related peptide (CGRP) suppresses the oxidative stress and vascular smooth muscle cell (VSMC) proliferation induced by vascular injury. A recent study also indicated that CGRP protects against the onset and development of angiotensin II (Ang II)-induced hypertension, vascular hypertrophy and oxidative stress. However, the mechanism behind the effects of CGRP on Ang II-induced oxidative stress is unclear. CGRP significantly suppressed the level of reactive oxygen species (ROS) generated by NADPH oxidase in Ang II-induced VSMCs. The Ang II-stimulated activation of both Src and the downstream transcription factor, STAT3, was abrogated by CGRP. However, the antioxidative effect of CGRP was lost following the expression of constitutively activated Src or STAT3. Pre-treatment with H-89 or CGRP_8-37 also blocked the CGRP inhibitory effects against Ang II-induced oxidative stress. Additionally, both in vitro and in vivo analyses show that CGRP treatment inhibited Ang II-induced VSMC proliferation and hypertrophy, accompanied by a reduction in ROS generation. Collectively, these results demonstrate that CGRP exhibits its antioxidative effect by blocking the Src/STAT3 signalling pathway that is associated with Ang II-induced VSMC hypertrophy and hyperplasia.

Keywords: Ang II; CGRP; ROS; STAT3; Src; VSMCs.

FIGURE 1

CGRP inhibited oxidative stress in Ang II‐induced VSMCs. Vascular smooth muscle cells (VSMCs), stimulated with CGRP (10⁻⁶‐10⁻⁸ mol/L) for 60 min, CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min or apocynin (10⁻⁶ mol/L) for 2 h, were treated with Ang II (10⁻⁷ mol/L) for 30 min. A, Quantification of intracellular reactive oxygen species (ROS) levels. ROS levels were measured using DCFH‐DA as described in method. B, Quantitation of the intracellular NADP/NADPH ratio. C, Western blot analysis of membrane and cytoplasmic fractions of p47phox in VSMCs. D, Quantitative real‐time PCR analysis of mRNA levels of p47phox in VSMCs. Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .01 and ***P < .001 vs Ang II. ^# P < .05 vs control

FIGURE 2

CGRP inhibited oxidative stress via receptors/ cAMP‐PKA‐dependent pathway. VSMCs, stimulated with CGRP (10⁻⁶‐10⁻⁸ mol/L) for 60 min or CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min, were treated with Ang II (10⁻⁷ mol/L) for 30 min. In some experiments, dibutyl‐cAMP (10⁻³ mol/L) was applied for 60 min and H‐89 was applied 30 min before CGRP pre‐treatment. A, Quantitative real‐time PCR analysis of the mRNA levels of CRLR, RAMP1 and RCP in VSMCs. B, Western blot analyses of protein levels of CRLR, RAMP1 and RCP in VSMCs. C, Quantification of intracellular ROS levels in VSMCs after pre‐treatment with dibutyl‐cAMP or H‐89. D, Quantitative real‐time PCR analysis of mRNA levels of p47phox in VSMCs. E, Western blot analysis of membrane and cytoplasmic fractions of p47phox in VSMCs. Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .01 and ***P < .001 vs Ang II. ^# P < .05 vs control. ⁺ P < .05 vs CGRP + Ang II

FIGURE 3

CGRP attenuated Ang II‐induced Src/STAT3 activation in VSMCs. VSMCs, stimulated with CGRP (10⁻⁷ mol/L) for 60 min or CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min, were treated with Ang II (10⁻⁷ mol/L) for 30 min. A and B, Phosphorylation of Src and STAT3 was evaluated via Western blot analysis. C and D, The distribution and levels of p‐Src and p‐STAT3 were detected via immunofluorescence. E, VSMCs were treated with PP2 (10⁻⁵ mol/L, 20 min), and phosphorylation of STAT3 was evaluated via Western blot analysis. Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .01 and ***P < .001 vs Ang II. ^# P < .05 vs control

FIGURE 4

CGRP attenuated Ang II‐induced Src/STAT3 activation via receptor/cAMP‐PKA‐dependent pathway in VSMCs. VSMCs, stimulated with CGRP (10⁻⁷ mol/L) for 60 min or CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min, were treated with Ang II (10⁻⁷ mol/L) for 30 min. In some experiments, dibutyl‐cAMP (10⁻³ mol/L) and H‐89 (10⁻⁵ mol/L) were applied 60 and 30 min before CGRP pre‐treatment, respectively. Phosphorylation of Src (A) and STAT3 (B) was evaluated via Western blot analysis. Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .01 and ***P < .001 vs Ang II. ^# P < .05 vs control

FIGURE 5

Src/STAT3 signalling pathway is involved in the antioxidant activity of CGRP. VSMCs, stimulated with CGRP (10⁻⁷ mol/L) for 60 min or CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min, were treated with Ang II (10⁻⁷ mol/L) for 30 min. In some experiments, PP2 (10⁻⁵ mol/L) or niclosamide (10⁻⁵ mol/L) was applied 20 min before CGRP pre‐treatment. VSMCs were transfected with Src ORF cDNA clones (Src) or STAT3 ORF cDNA clones (STAT3) and incubated for 24 h. Increased Src (A) and STAT3 (B) protein levels in VSMCs were evaluated via Western blot analysis after transfection with Src or STAT3 ORF cDNA clones, respectively. (C), Quantification of intracellular ROS levels in VSMCs via DCFH‐DA. Src group, VSMCs were transfected with Src ORF cDNA clones; STAT3 group, VSMCs were transfected with STAT3 ORF cDNA clones; and control group, VSMCs were transfected with blank vector. Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .01 and ***P < .001 vs Ang II. ^# P < .05 vs control. ⁺ P < .05 vs CGRP + Ang II

FIGURE 6

CGRP suppressed hypertrophy and hyperplasia of VSMCs in vitro and in vivo. VSMCs, stimulated with CGRP (10⁻⁷ mol/L) for 60 min or CGRP_8‐37 (3 × 10⁻⁵ mol/L) for 30 min, were treated with Ang II (10⁻⁷ mol/L) for 30 min. In some experiments, NAC (10⁻² mol/L) or H₂O₂ (10⁻³ mol/L) was applied 30 min before CGRP or Ang II pre‐treatment. A and B, The proliferation of VSMCs was analysed using BrdU assays. C, Systolic blood pressure (SBP) was measured by tail‐cuff plethysmography in three groups: control group (saline‐treated), Ang II (750 µg/kg/d, in saline)‐treated group and Ang II + CGRP (50 nmol/d, in saline)‐treated group (n = 5). D, The medial thickness of the abdominal aortas was assayed in the Ang II‐treated and Ang II + CGRP‐treated groups (n = 5). E, The 8‐OHdG immunohistochemistry of the abdominal aortas in the Ang II‐treated and Ang II + CGRP‐treated groups (n = 5). Bar graphs show mean ± SEM values from three independent experiments. *P < .05, **P < .05 vs Ang Ⅱ. ^# P < .05 vs control