Pulmonary, Gastrointestinal and Urogenital PharmacologyInhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: Role of ERK1/2 and p27
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).
References (33)
- et al.
Role of calcitonin gene-related peptide (CGRP) in chronic hypoxia-induced pulmonary hypertension in the mouse. Influence of gene transfer in vivo
Regul. Pept.
(2002) - et al.
Oxidant-mediated activation of mitogen-activated protein kinases and nuclear transcription factors in the cardiovascular system: a brief overview
Cell. Signal.
(1998) - et al.
The cell cycle: a critical therapeutic target to prevent vascular proliferative disease
Can. J. Cardiol.
(2006) - et al.
Inhibition of vascular smooth muscle cell proliferation in vitro by genetically engineered marrow stromal cells secreting calcitonin gene-related peptide
Life Sci.
(2006) - et al.
Involvement of asymmetric dimethylarginine and Rho kinase in the vascular remodeling in monocrotaline-induced pulmonary hypertension
Vascul. Pharmacol.
(2010) - et al.
Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells
Eur. J. Pharmacol.
(2004) - et al.
Vascular remodeling in pulmonary arterial hypertension: multiple cancer-like pathways and possible treatment modalities
Int. J. Cardiol.
(2011) - et al.
Calcitonin gene-related peptide (CGRP) and its role in hypertension
Neuropeptides
(2011) - et al.
Effect of prepro-calcitoningene-related peptide-expressing endothelial progenitor cells on pulmonary hypertension
Ann. Thorac. Surg.
(2007) - et al.
Krüppel-like factor 4 inhibits proliferation by platelet-derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha-mediated, phosphatidylinositol 3-kinase and ERK signaling in vascular smooth muscle cells
J. Biol. Chem.
(2009)
Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target
Circulation
In vivo gene transfer of prepro-calcitonin gene-related peptide to the lung attenuates chronic hypoxia-induced pulmonary hypertension in the mouse
Circulation
Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells
Am. J. Physiol. Lung Cell. Mol. Physiol.
Role of epidermal growth factor inhibition in experimental pulmonary hypertension
Am. J. Respir. Crit. Care Med.
Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFR beta-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia
Cell Res.
Regulation of the cell cycle inhibitor p27 and its ubiquitin ligase Skp2 in differentiation of human embryonic stem cells
FASEB J.
Cited by (38)
Protective effects of intermedin/adrenomedullin-2 in a cellular model of human pulmonary arterial hypertension
2020, PeptidesCitation Excerpt :The two most common rodent models, the chronic hypoxic model and the monocrotaline injury model, have contributed to understanding of the human PAH process but are not without limitations [33]. CGRP and AM have been found to attenuate pulmonary artery pressure and vascular remodelling in such models [34,35]; a vasorelaxant effect of IMD/AM2 has also recently been reported in hypoxia-induced pulmonary hypertensive rat [36]. Evidence for receptor subtype and endothelium dependence/nitric oxide involvement in these relaxant responses is conflicting [37,38].
Osthole inhibits cell proliferation by regulating the TGF-β1/Smad/p38 signaling pathways in pulmonary arterial smooth muscle cells
2020, Biomedicine and PharmacotherapySUMOylation of Vps34 by SUMO1 promotes phenotypic switching of vascular smooth muscle cells by activating autophagy in pulmonary arterial hypertension
2019, Pulmonary Pharmacology and TherapeuticsCitation Excerpt :The ratio of RV/(LV + Septum) was used as an indicator of right ventricle (RV) hypertrophy. The percentage of wall thickness of the media was measured as previously described [49,50]. The percentage of vascular media wall thickness was calculated by the formula: vascular media wall thickness (%) = area ext – area int/area ext × 100.
LOX-1 mediated phenotypic switching of pulmonary arterial smooth muscle cells contributes to hypoxic pulmonary hypertension
2018, European Journal of PharmacologyCitation Excerpt :HE and IHC staining of right lung were detected in accordance to the same method used in our previous studies (Li et al., 2012; Zhang et al., 2017b). Primary rat PASMCs were prepared from the pulmonary arteries of male SD rat (180–220 g) using explant method and identified according to the same method used in our previous studies (Li et al., 2012; Zhang et al., 2017b). The cells were cultured at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS).