Involvement Of Calcium-sensing Receptors In Hypoxia-induced Vascular Remodeling And Pulmonary Hypertension By Promoting Phenotypic Modulation Of Small Pulmonary Arteries

Background and Purpose Pulmonary arterial hypertension(PAH)is a progressive disease associated with a poor prognosis and potentially leads to heart dysfunction.Currently,there are limited options available for the prevention and treatment of progressive PAH.Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling and PAH development.Chronic hypoxia-induced PAH is characterized by increased right ventricular afterload from increased pulmonary vascular resistance due to pulmonary vasoconstriction and pulmonary vascular remodeling.Pulmonary vascular remodeling is associated with vascular smooth muscle cell(VSMC)phenotypic modulation.Phenotype modulation of pulmonary artery smooth muscle cells(PASMCs)plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension(PAH).We had previously shown that calcium-sensing receptor(Ca SR)is expressed in rat PASMCs.However,little is known about the role of Ca SR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms.In this study,we investigated whether Ca SR involves the proliferation and phenotypic modulation of PASMCs in small pulmonary arteries from both rats and human with PAH.Methods(1)PAH was induced by exposing rats to hypoxia for 7–21 days.(2)The mean pulmonary arterial pressure(m PAP),right ventricular hypertrophy index(RVI),the percentage of medial wall thickness to the external diameter(WT %),and cross-sectional total vessel wall area to the total area(WA %)of small pulmonary arteries were determined by hematoxylin and eosin(HE),masson trichrome and Weigert's staining.(3)The protein expressions of matrix metalloproteinase(MMP)-2 and MMP-9,the tissue inhibitors of metalloproteinase(TIMP)-3,Ca SR,proliferating cell nuclear antigen(PCNA)and smooth muscle cell(SMC)phenotype marker proteins in rat small pulmonary arteries,including calponin,SMA-actin(SMAa)and osteopontin(OPN)were analyzed by immunohistochemistry and Western blotting,respectively.(4)Angiotasis of pulmonary artery was detected using the tissue chamber vascular ring technique.(5)In addition,immunohistochemistry was applied to paraffinembedded human tissues from lungs of normal human and PAH patients with chronic heart failure(PAH/CHF).(6)PAMSCs were incubated 24 hour to establish a model of hypoxia.At the same time pretreated 24 hour with R568,NPS 2390,SB203580 and LY294002.Then,analyzed the expression of Ca SR?PCNA?Ki67?phenotype marker proteins?MMP-2?MMP-9?TIMP-3?p-p38 MAPK and p-Akt using Western blot.Results In vivo(1)Compared with the control group,m PAP,RVI,WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia.(2)At the same time,the expressions of Ca SR,MMP-2,MMP-9,TIMP-3,PCNA and OPN were markedly increased,while the expressions of SMA? and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats.(3)Compared with the control group,the tension of pulmonary loop was significantly increased in hypoxia 9d group.(4)Neomycin(anagonist of Ca SR)enhanced but NPS2390(an antagonist of Ca SR)weakened these hypoxic effects.In vitro After hypoxia 24 hour,the expressions of Ca SR,PCNA,Ki67,OPN were significantly increased and calponin,SMA-? were decreased in the PASMCs,and the protein of p38 MAPK and Akt were phosphorylated.R568 can amplify these effects.However inhibitor of the p38 MAPK signal pathway(SB203580)and inhibitor of the PI3K/PKB(Akt)signal pathway(LY294002)can decrease these roles.Clinical specimen Compared with normal human lungs,the expressions of Ca SR,PCNA and SMC phenotypic marker proteins in PAH/CHF lungs were significantly increased.Conclusions Our data suggest that Ca SR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.(1)The expression of Ca SR in hypoxic rat PAH is gradually increased and contributes to media wall thickening which via participated in the phenotypic modulation of PASMCs from contractile to synthetic-type.(2)Ca SR is involved in Pulmonary vascontriction and vascular remodeling of hypoxic induced PAH,which may be associated with the activation of p38 MAPK,PI3K/Akt signaling pathway.