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 CGRP8–37 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.

are involved in mediating the signal transduction of Ang II-induced hypertrophy. 4 All vascular cell types, including endothelial cells and VSMCs, are capable of producing ROS. Ang II-stimulated ROS are generated in VSMCs through the activity of NADPH oxidase, which contains five components (p47phox, p67phox, p40phox, p22phox and gp91phox). 5 During this Ang II-mediated activation of the enzyme, the phosphorylation of p47phox, in particular, is critical, as it triggers the formation of a complex between p47phox and ROS production-related molecules in the cytoplasm, enhancing their translocation to the membrane. 6 The calcitonin gene-related peptide (CGRP), which is composed of 37 amino acids and produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene, is a potent vasodilator and hypotensive peptide. 7 CGRP elicits its biological actions via non-selective interaction with the calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 1 (RAMP1) and the intracellular receptor component protein (RCP), following the activation of the cAMP/protein kinase A (PKA)-dependent pathway. 8 CGRP has been shown to exert various effects within the cardiovascular system, including the abrogation of antioxidative stress and inhibition of VSMC proliferation and migration. 9 Recently, we reported that endogenous CGRP suppresses VSMC proliferation and oxidative stress induced by vascular injury. 10 In addition, CGRP protects against the onset and development of Ang II-induced hypertension, vascular hypertrophy and oxidative stress. 11 These data confirmed the hypothesis that CGRP plays a protective role against Ang II-induced oxidative stress in VSMCs, and the antioxidative effect may be associated with the pathophysiological effects on these muscle cells. However, the underlying cellular mechanisms involved are still unclear, and the critical CGRP-regulated signal transduction pathways are yet to be identified.
Ang II binding to the signalling molecules that modulate ROS production in vascular cells is a complex mechanism that may occur at the transcriptional or post-transcriptional level, involving the generation of several intermediate signalling molecules. 4 Src, belonging to the family of non-receptor tyrosine kinases, is one such signalling molecule. Src regulates several cellular pathways by phosphorylating the proteins involved in the mechanical and chemical stimulation processes that modulate ROS generation in VSMCs 12 or regulate VSMC proliferation and migration in response to interaction with Ang II. 13 Several studies have revealed numerous intracellular events occurring from the point of Src kinase activation to the expression of downstream target proteins, such as the signal transducer and activator of transcription 3 (STAT3). [14][15][16] Src mediates the activation of STAT3, which subsequently regulates various VSMC processes, including cell proliferation and migration. 17 Additionally, the roles of STAT3 in regulating ROS production and oxidative metabolism have recently been highlighted. 18 However, very little is known about the association between Ang II and the Src/STAT3 signalling pathway modulating ROS production in VSMCs. As adrenomedullin, a member of the calcitonin peptide superfamily, inhibits Ang II-induced oxidative stress via the Csk-mediated inhibition of Src activity, 19 we have been suggested that the Src/STAT3 signalling pathway would be a potential pathway through which CGRP interrupts Ang II-induced ROS production. Therefore, in this study, we investigated whether the Src/STAT3 signalling pathway is associated with the antioxidative effect of CGRP and whether it consequently prevents Ang II-induced hypertrophy and hyperplasia of VSMCs in vitro and in vivo.

| VSMCs
Rat VSMCs were isolated from the aorta of 80-100 g male Sprague-Dawley rats and cut into small pieces as previously described. 10 The tunica medium was separated from the adventitia and endothelium and cultured in DMEM (Invitrogen) supplemented with 10% FBS and a mixture of 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen). α-actin testing of cultured cells confirmed a positive response. The VSMCs were maintained at 37°C in a humidified atmosphere containing 5% CO 2 , and only passage 3 to passage 5 cells at 70%-80% confluence were used in the experiments, except if stated otherwise. Each individual experiment was repeated at least thrice with different cell preparations.

| Histology and immunohistochemistry
The abdominal aortas were excised from each mouse, fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. The aortas were then cut into 5-μm sections, which were stained with haematoxylin and eosin (HE) and Elastica Van Gieson (EVG). For immunohistochemical analysis, arterial sections were incubated with rabbit anti-8-OHdG (1:100, Bioss, catalogue # bs-1278R). The analysis software BZ-II analyzer (Keyence) was applied to acquire the immunofluorescence average optical density (AOD) for evaluating the expression level of p-Src and p-STAT3 in VSMCs.

| NADP/NADPH assay
Quantitation of the intracellular NADP/NADPH ratio was measured using the Amplite Fluorimetric NADP/NADPH assay kit (AAT Bioquest, Inc), according to the manufacturer's instructions.
Briefly, cells were pre-treated with or without test compounds for the indicated time periods and then stimulated with or without Ang II (10 −7 mol/L) for 30 minutes and CGRP (10 −6 -10 −8 mol/L) for 60 minutes. In some experiments, CGRP  or H-89 was added 30 minutes, dibutyl-cAMP 60 minutes and apocynin (10 −6 mol/L) 2 hours before CGRP treatment. NADP/NADPH ratio measurements were determined based on an enzymatic cycling reaction, using a Microplate reader (excitation: 540 nm and emission: 590 nm).

| cAMP and PKA measurement
VSMCs were first pre-treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8-37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers' protocols.

| Transient transfection
For the transfection, VSMCs were cultured on a 6-well plate at a density of 1 × 10 5 cells/well for 24 hours. Then, the cells were transfected with 150 ng of Src ORF cDNA, STAT3 ORF cDNA or blank pReceiver-M13 vector (control) for 48 hours, and the transfection efficiency was confirmed via immunoblotting for total protein. To determine p47phox translocation, the membrane and cytosolic fraction were separated by ultracentrifugation. p47phox

| Western blot analysis
(1:100) abundance was also assessed in membrane and cytoplasmic fractions.

| Quantitative real-time PCR
After appropriate treatments, total RNA from VSMCs was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The cDNA was synthesized using reverse transcriptase (Invitrogen). Real-time PCR was carried out using an Applied Biosystems 7500 fast PCR System (Life Technologies, USA) and SYBR Green RT-PCR Kit (Invitrogen). Values were normalized to rat GAPDH. Oligonucleotide primers for real-time PCR amplification (Table 1) were synthesized by Sangon Biotech (Shanghai).

| Cell proliferation assays
VSMCs (1 × 10 5 ) were cultured in 96-well plates, incubated in DMEM (Invitrogen) without serum for 16 hours and then treated with CGRP, Ang II or NAC for 12 hours. VSMC proliferation was then meas-

| Blood pressure evaluation
Systolic blood pressure was measured on days 0 (basal), 7 and 14 before pump implantation and every 3-6 days after implantation using a standard tail cuff (BP-2010A System, Softron).

| Statistical analysis
Values are expressed as means ± SEM. Student's t test was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences between three or more groups. Values of P < .05 were considered significant.

| CGRP inhibited oxidative stress in Ang IIinduced VSMCs
In this study, we first examined the effects of CGRP on the oxidative stress stimulated by Ang II in VSMCs. CGRP significantly suppressed ROS production in Ang II-stimulated VSMCs in a time-dependent ( Figure S1A) and dose-dependent ( Figure 1A) manner. It is well known that the Ang II-stimulated activation of NADPH oxidase, especially the p47phox subunit, results in ROS generation in VSMCs.
We therefore evaluated NADPH oxidase activation and found that CGRP decreased the intracellular NADP/NADPH ratio ( Figure 1B).
The p47phox amount was reduced in the cytoplasmic fraction and increased in the membrane fraction after Ang II stimulation, but these effects were reversed in the CGRP-pre-treated VSMCs ( Figure 1C).
Consistently, CGRP treatment significantly decreased the p47phox mRNA levels in Ang II-induced VSMCs ( Figure 1D). Additionally, CGRP inhibited ROS generation in VSMCs to almost the same level as that observed after treatment of the cells with an NADPH oxidase-specific inhibitor, apocynin ( Figure 1A). These data suggest that CGRP inhibited Ang II-induced oxidative stress in VSMCs by inhibiting both NADPH oxidase activation and ROS production.

| CGRP inhibited oxidative stress via receptors and the cAMP/PKA-dependent pathway
CGRP exerts its biological effects by activating the cAMP/PKAdependent signalling pathway by binding to its receptors, the CRLR/ RAMP1/RCP system. To identify the role of the CGRP receptor-dependent signalling pathway in inhibiting Ang II-stimulated oxidative stress in VSMCs, we first examined the changes in CGRP receptors following Ang II induction. Both the mRNA and protein levels of CRLR increased in VSMCs after Ang II stimulation, whereas RAMP1 and RCP levels decreased significantly. However, these changes in the receptor levels were reversed by CGRP stimulation in a dosedependent manner (Figure 2A,B).
In VSMCs pre-treated with a CGRP receptor antagonist (CGRP 8-37 ), the CGRP-mediated inhibition of Ang II-induced oxidative stress was completely abolished, including the inhibition of p47phox activation and ROS production ( Figure 1). We further examined the involvement of the cAMP/PKA-dependent pathway in the CGRP inhibition of oxidative stress generation. The cAMP and PKA concentrations in Ang II-stimulated VSMCs increased after CGRP stimulation, and the effect was again blocked by CGRP 8-37 treatment ( Figure S2A,B). Pre-treatment of the VSMCs with dibutyl-cAMP further promoted the inhibitory effect of CGRP on ROS generation, whereas pre-treatment with H-89 (a PKA inhibitor) completely abrogated the inhibitory effect ( Figure 2C). We then examined the mRNA levels of p47phox, and its translocation in Ang II-stimulated and CGRP-treated cells, and found that dibutyl-cAMP and H-89 treatments had similar effects ( Figure 2D,E).

| CGRP attenuated Ang II-induced Src/STAT3 activation in VSMCs
Both Src and STAT3 have been suggested to be involved in the regulation of Ang II-induced VSMC responses. Therefore, the role of the Src/ STAT3 signalling pathway was examined. As shown in Figure 3A Figure 3E). The results showed that CGRP inhibition of Ang II-induced STAT3 activation was associated with changes in the Src activity.

| CGRP attenuated Ang II-induced Src/STAT3 activation via the receptor/cAMP/PKA-dependent pathway in VSMCs
It is becoming clear that the Src and STAT3 pathways are potential effector pathways for G proteins and may play a role in G protein function. Therefore, we investigated whether CGRP attenuated Src/STAT3 activation through a G protein-coupled receptor (GPCR)-mediated signalling cascade, that is via the cAMP/PKA-dependent pathway. Figure 4A,B shows the effects of dibutyl-cAMP, H-89 and CGRP 8-37 treatment on Src and STAT3 phosphorylation. We found that dibutyl-cAMP further promoted the inhibitory effect of CGRP on Src and STAT3 phosphorylation, whereas H-89 treatment completely abolished this inhibitory effect. Ang II (Figure 5C).

| CGRP suppressed the hypertrophy and hyperplasia of VSMCs in vitro and in vivo
To ascertain whether CGRP suppresses the vascular hypertrophy and hyperplasia of VSMCs by inhibiting oxidative stress, we first Then, we infused mice with Ang II for 14 days to examine the effects of CGRP on Ang II-induced vascular hypertrophy. As CGRP has been reported to lower blood pressure, we verified this effect in the Ang II-infused mice. As expected, Ang II infusion increased the systolic blood pressure (SBP) of the mice, whereas hypertension was significantly reversed in CGRP-treated mice ( Figure 6C). HE and EVG staining showed that CGRP treatment significantly inhibited Ang IIinduced thickening of the abdominal aortas ( Figure 6D). In addition, the immunostaining assay for 8-OHdG, a marker for ROS-induced DNA damage, showed that CGRP treatment had decreased the level of ROS damage induced by Ang II relative to that in the untreated mice ( Figure 6E). Taken together, these in vitro and in vivo results suggest that CGRP may play key roles in the Ang II-induced hypertrophy and hyperplasia of VSMCs by suppressing oxidative stress.

| D ISCUSS I ON
The CGRP has been reported to elicit protective effects against cell injuries in several disease models, where the mechanisms underlying its protective effects differed depending on the cell types and experimental conditions. 11,20-23 We had previously reported that CGRP deficiency led to enhanced neointima formation after vascular injury, where the cells also showed a higher degree of oxidative stress. 10 In the present study, we found that CGRP inhibited Ang II-induced oxidative stress, both in vitro and in vivo, including the inhibition of NADPH oxidase activation, p47phox activation and ROS production.
As NADPH oxidase is an important enzyme for ROS production, we determined whether CGRP reduced ROS production by inhibiting NADPH oxidase, using apocynin (an NADPH oxidase inhibitor) as a positive control for the in vivo study. We found that both CGRP and apocynin reduced Ang II-stimulated ROS generation to a similar degree. Thus, CGRP may reduce the ROS generated in Ang II-induced VSMCs by inhibiting NADPH oxidase directly.
CGRP is involved in the regulation of the cardiovascular system via its receptors. For instance, in an animal model of Ang II-induced hypertension, RAMP1 transgenic mice were shown to be protected from increases in their SBP. 24 However, the CGRP and/or CGRP receptor expression profile varies with the state and stage of the disease, including that in the Ang II-induced model. 25,26 In addition, the change in CGRP receptor expression In view of these results, it appears that CGRP may act as a regulatory autocrine or paracrine factor via its receptors to exert its functions in Ang II-induced VSMCs.
The intracellular signalling mediated by CGRP and its receptors is highly complex. The cAMP/PKA-dependent pathway is the primary signalling pathway involved in the CGRP effect on the vasculature, especially in VSMCs. 28 We observed that CGRP increased the intracellular cAMP content and PKA activity in Ang In summary, our results demonstrate that CGRP exhibits its antioxidative effect by blocking the Src/STAT3 signalling pathway, which is associated with the hypertrophy and hyperplasia of VSMCs induced by Ang II. Moreover, the current study provides molecular F I G U R E 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 H 2 O 2 (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 evidence in support of previous reports on the important role of CGRP in protecting against Ang II-induced oxidative stress in VSMCs during the development of hypertension.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Nature Science

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support our findings of this study are available on request from the corresponding author.