Pulmonary, Gastrointestinal and Urogenital Pharmacology
Inhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: Role of ERK1/2 and p27

https://doi.org/10.1016/j.ejphar.2012.01.015Get rights and content

Abstract

Calcitonin gene-related peptide (CGRP) inhibits angiotensin II-induced proliferation of aortic smooth muscle cells via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 is necessary for the degradation or down-regulation of the cell cycle inhibitor p27, and is also crucial in mediating proliferation of pulmonary artery smooth muscle cells (PASMCs). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown. Pulmonary hypertension was induced by hypoxia in rats, and capsaicin (50 mg/kg, s.c.) was used to deplete endogenous CGRP. Proliferation of cultured PASMCs was determined by BrdU incorporation method and flow cytometry. The expression/level of CGRP, p27, ERK1/2, c-fos and c-myc was analyzed by radioimmunoassay, immunohistochemistry, real-time PCR or Western blot. Sensory CGRP depletion by capsaicin exacerbated hypoxia-induced pulmonary hypertension in rats, as shown by an increase in right ventricle systolic pressure, mean pulmonary artery pressure and vascular hypertrophy, accompanied with decreased p27 expression and increased expression of phosphorylated ERK1/2, c-fos and c-myc. Exogenous application of CGRP significantly inhibited hypoxia-induced proliferation of PASMCs concomitantly with increased p27 expression and decreased expression of phosphorylated ERK1/2, c-fos and c-myc. These effects of CGRP were abolished in the presence of CGRP8–37. Knockdown of p27 also reversed the inhibitory effect of CGRP on proliferation of PASMCs and expression of c-fos and c-myc, but not on ERK1/2 phosphorylation. These results suggest that CGRP inhibits hypoxia-induced proliferation of PASMCs via ERK1/2/p27/c-fos/c-myc pathway. Down-regulation of CGRP may contribute to remodeling of pulmonary arteries in hypoxia-induced pulmonary hypertension.

Introduction

Pulmonary hypertension is a syndrome in which obstructed, constricted small pulmonary arteries and increased pulmonary vascular resistance ultimately leading to right ventricular hypertrophy and failure. Although the pathogenesis of pulmonary hypertension has not been fully understood, it is well accepted that vascular remodeling is a hallmark of pulmonary hypertension, and the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) play a major role in vascular remodeling (Dahal et al., 2010, Lykouras et al., 2008). In normal situation, most of PASMCs in healthy adult are in a quiescent state, while proliferative PASMCs are found in pulmonary arterioles of patients with pulmonary hypertension (Archer et al., 2010, Prabha et al., 2008).

The balance between cell proliferation and cell quiescence is regulated by a variety of cell cycle modulators. Cyclin-dependent kinase (CDK) is a major regulator of the transition between the phases of the cell cycle (Yu et al., 2005). Cyclin/CDK complexes are composed of a regulatory subunit, cyclin, and an active kinase subunit, CDK. The cyclin/CDK complexes are controlled by both positive and negative regulators (Yu et al., 2005). p27, a member of the Cip/Kip family of CDK inhibitors, is a negative regulator of the protein kinase CDK2/cyclin E which can block the cell cycle at G0/G1 phase (Egozi et al., 2007). The levels of p27 are high in the G0/G1 phases of cell cycle. Upon mitogenic stimulation, p27 is rapidly degraded, thus allowing the action of CDK2/cyclin E to promote cell proliferation (Lightell et al., 2011). It has been found that the expression of p27 in serum-stimulated vascular smooth muscle cells (VSMCs) inactivates CDK2 and CDK4, leading to G1 growth arrest (Tanner et al., 2000). In an in vivo model of vascular injury, overexpression of p27 also reduces proliferation of intimal VSMCs and the intima/media area ratio (Tanner et al., 2000). In vitro, loss of the p27 gene negates the inhibitory effect of heparin on proliferation of PASMCs (Yu et al., 2005). In vivo, heparin significantly inhibits the development of hypoxia-induced pulmonary hypertension and vascular remodeling in wild type mice, but not in p27 null mice (Yu et al., 2005). These finding suggest that cell cycle inhibition afforded by p27 holds great potential as a therapeutic strategy for vascular proliferative diseases (Charron et al., 2006, Vokurkova et al., 2007).

Calcitonin gene-related peptide (CGRP), a 37-amino acid endogenous sensory nerve peptide, is widely distributed throughout the central and peripheral nervous systems. In the cardiovascular system, CGRP-containing nerves are located throughout the heart and lung and surround most arteries. CGRP has a broad range of activities, including acting as a sensory neuropeptide and a potent vasodilator. A growing of evidence shows that endogenous CGRP plays a crucial role in pathogenesis of pulmonary hypertension and vascular remodeling (Tjen et al., 1998, Keith et al., 2000). In vivo, adenovirus-mediated over-expression of CGRP increases expression of p53 to stimulate p21, which inhibits cyclin/CDK complexes, thereby inhibiting cell proliferation of PASMCs (Chattergoon et al., 2005). Our previous study has found that CGRP inhibits proliferation of aortic smooth muscle cells induced by angiotensin II via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) signal pathway (Qin et al., 2004). It has been shown that ERK1/2 signal pathway is necessary for the degradation or down-regulation of p27, and is also crucial in mediating proliferation of PASMCs in response to a multitude of mitogenic stimuli and vascular injury (Dong et al., 2010, Perez et al., 2011, Zheng et al., 2009). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown.

By using models of hypoxia-induced rat pulmonary hypertension and proliferation of PASMCs, we performed the present study with two related hypotheses. First, we analyzed the correlation of ERK1/2/p27 signal pathway and CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling in rats. Second, as proliferation of PASMCs plays a vital role in pulmonary vascular remodeling, we evaluated the role of ERK1/2/p27 signal pathway in mediating the inhibitory effect of CGRP on hypoxia-induced proliferation of rat PASMCs.

Section snippets

Animals

Male Sprague–Dawley rats weighing 180–220 g were obtained from Laboratory Animal Center, Xiangya School of Medicine, Central South University (Changsha, China). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Medicine Animal Welfare Committee of Xiangya School of Medicine, Central South University.

Animal experiments

Rats were randomly divided into three groups: (i) Normoxia group;

Effect of capsaicin on cardiovascular remodeling in hypoxia-induced pulmonary hypertension rats

In keeping with previous study (Tjen et al., 1998), three weeks of exposure to hypoxia induced pulmonary hypertension in rats, as shown by a significant elevation in RVSP and mPAP compared with the normoxic rats (Fig. 1A and B). Hypoxia also significantly induced hypertrophy of right ventricle and pulmonary arteries, showing an increase in ratio of RV/LV + S, percent of WT and proliferation of smooth muscle cells in the vascular media of small pulmonary arteries compared with normoxic control (

Discussion

It has been demonstrated that three-week neonatal hypoxia reduces blood CGRP level and causes persistent pulmonary hypertension in rats (Keith et al., 2000). Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic adult rats (Qing and Keith, 2003). Our present study confirmed the previous observation that sensory CGRP depletion by capsaicin exacerbates hypoxia-induced pulmonary hypertension in adult rats (Tjen et al., 1998). Interestingly, in vivo

Acknowledgments

This work was supported by a grant from the National Natural Science Fund of China (No. 81072638).

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