Calcitonin gene-related peptide attenuates angiotensin II-induced ROS-dependent apoptosis in vascular smooth muscle cells by inhibiting the CaMKII/CREB signalling pathway

https://doi.org/10.1016/j.bbrc.2019.10.064Get rights and content

Highlights

  • CGRP inhibited Ang II-induced apoptosis in VSMCs.

  • CGRP attenuated Ang II-induced CaMKII/CREB signaling pathway in VSMCs.

  • The anti-apoptotic effect of CGRP might be mediated by inhibiting the ROS-dependent CaMKII/CREB pathways.

Abstract

Apoptosis is associated with various cardiovascular diseases. CGRP exerts a variety of effects within the cardiovascular system, and protects against the onset and development of angiotensin (Ang) II-induced vascular dysfunction and remodelling. However, it is not known whether CGRP has a direct effect on Ang II-induced apoptosis in vascular smooth muscle cells (VSMCs), and the mechanism underlying the anti-apoptotic role remains unclear. In this study, CGRP significantly suppressed reactive oxygen species (ROS) and apoptosis in Ang II-induced VSMCs. In VSMCs pre-treated with a CGRP receptor antagonist (CGRP8–37), the CGRP-mediated inhibition of Ang II-induced ROS and apoptosis was completely abolished. Moreover, pre-treatment with N-acetyl-L cysteine (NAC), an ROS scavenger, blocked the effects of CGRP on Ang II-induced apoptosis. In addition, the activation of CaMKII and the downstream transcription factor CREB stimulated by Ang II was abrogated by CGRP. Importantly, in both CGRP and NAC-treated VSMCs, CGRP failed to further attenuate CaMKII and CREB activation. The results demonstrate that CGRP attenuated Ang II-induced ROS-dependent apoptosis in VSMCs by inhibiting the CaMKII/CREB signalling pathway.

Introduction

Accumulating evidence has shown that apoptosis of vascular smooth muscle cells (VSMCs) is a key event underlying many VSMC-related pathological conditions, such as hypertension and atherosclerosis [1,2]. Angiotensin II (Ang II), the primary effector molecule of the renin-angiotensin system, plays a major role in the regulation of vascular function and structure [3,4]. Ang II can increase arterial pressure and induce VSMC hypertrophy and apoptosis. The apoptotic effects of Ang II are mediated by a number of intracellular signalling pathways and molecules including PLC, Ca2+ channels, PLD, PLA2, adenylate cyclase, MAP kinases, the JAK-STAT pathway, and reactive oxygen species (ROS) [5,6].

The trophic and contractile actions of Ang II are dependent on the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is known to regulate cyclic AMP response element-binding protein (CREB) [7], and together they are involved in the regulation of VSMC proliferation, migration, inflammation, and apoptosis [8,9]. CaMKII is a ubiquitous kinase that is not only activated by an elevation in intracellular Ca2+, but is also modulated by ROS-dependent oxidation [10]. ROS, which participate in the pathogenesis of cardiovascular diseases, are involved in mediating the signal transduction of Ang II-induced hypertrophy [11]. In addition, Ang II-induced apoptosis is caused by an increase in CaMKII activity mediated by ROS [4,10]. Thus, it is plausible that CaMKII/CREB may be activated downstream of ROS signalling by Ang II in VSMCs, which likely mediates Ang II-induced VSMC apoptosis.

The calcitonin gene-related peptide (CGRP), 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 [12]. CGRP elicits its biological actions via non-selective interaction with different combinations of calcitonin receptor-like receptor, receptor activity modifying protein 1, and an intracellular protein, receptor component protein (RCP), which activate the cAMP/protein kinase A pathway [13]. CGRP has been shown to exert a variety of effects within the cardiovascular system, and protects against the onset and development of Ang II-induced vascular dysfunction and remodelling [14,15]. Recently, we reported that endogenous CGRP suppresses VSMC proliferation and oxidative stress induced by vascular injury [16]. In addition, CGRP partly protects VMSCs against H2O2-induced apoptosis [17]. These data indicate that CGRP protects against Ang II-induced injuries in VSMCs. However, it is not known whether CGRP has a direct effect on Ang II-induced apoptosis in VSMCs, and the mechanism underlying the anti-apoptotic role remains unclear. Since CGRP regulates neuronal CaMKII/CREB cascades mediating the development of tolerance to morphine-induced analgesia [18], we hypothesized that the CaMKII/CREB signalling pathway is a potential candidate pathway through which CGRP intersects with Ang II-induced apoptosis. Therefore, in this study, we investigated whether a possible crosstalk between ROS and the CaMKII/CREB signalling pathway participated in the anti-apoptotic effect of CGRP in VSMCs in response to Ang II.

Section snippets

Materials

Human CGRP, human CGRP8–37, Ang II, N-acetyl-L cysteine (NAC), and KN93 were purchased from Sigma. (Cupertino, CA, USA). Anti-phospho-CaMKII (Tyr286), anti-phospho-CREB (Ser133), anti-CaMKII, and anti-CREB antibodies were purchased from Cell Signalling Technology Inc. (Danvers, MA, USA). Anti-caspase-3 was purchased from Abcam. GAPDH and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-BCL-2 (AF6139) and anti-Bax (AF0120) were purchased from Affinity

CGRP inhibited Ang II-induced apoptosis in VSMCs

In this study, we first examined the effects of CGRP on Ang II-induced apoptosis in VSMCs. Flow cytometry results revealed that a marked increase in the apoptosis rate was observed in VSMCs that had been incubated with Ang II, and CGRP treatment markedly reduced Ang II-induced cell apoptosis (Fig. 1A and B). In addition, we found that CGRP decreased the expression levels of the pro-apoptotic protein Bax and caspase-3 and increased the expression levels of the anti-apoptotic protein Bcl-2 (Fig. 1

Discussion

In this study, CGRP suppressed apoptosis in Ang II-induced VSMCs, and the anti-apoptotic effect of CGRP may be mediated by inhibiting the ROS-dependent CaMKII/CREB pathways.

VSMCs in human plaques or derived from plaques show increased susceptibility to apoptosis, and VSMC apoptosis is a primary and early event in cardiovascular diseases [19]. CGRP has been reported to elicit protective effects against injuries in several disease models, and the mechanisms underlying its protective effects

Sources of funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81600563 and 81400322), Science and Technology Research Project of Colleges and Universities in Hebei Province (No. QN2014005), Hebei Medical Science Research Project (No. 20170125, 20150631 and 20180414).

Declaration of competing interest

The authors declare that there are no competing interests associated with the present study.

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