Role And Mechanism Of Carnitine Palmitoyltransferase 1 In Vascular Remodeling Of Pulmonary Arterial Hypertension In Rats

BackgroundPulmonary arterial hypertension?PAH?is a disease with increasing pulmonary vascular resistance,gradually increased pulmonary artery pressure,and the prognosis is poor.PAH animal induced by intraperitoneal injection of monocrotaline?MCT?is a classic model for the PAH mechanism and treatment studies.PAH in human manifests as a chronic process.However,PAH induced by single injection of relatively high dose of MCT in animal is a subacute process,with a short survival period less than 1 month.It is necessary to establish a new PAH model to improve survival duration and rate.The main pathological changes of PAH include pulmonary vascular remodeling and elevated pulmonary vascular tone,in which the abnormal proliferation and migration of PASMCs?pulmonary artery smooth muscle cells?play a dominant role.Unfortunately,the mechanism of PASMCs proliferation is so complicated that it has not been fully elucidated.Some studies and our previous work have found that during the development of PAH,fatty acid metabolism was abnormal.We have also found that CPT?carnitine palmitoyltransferase?1,the key rate-limiting enzyme of mitochondrial fatty acid beta-oxidation,was upregulated in lungs of PAH rats.However,the role and mechanism of CPT1 in the PASMCs proliferation,migration and development of PAH is still unknown.Objectives1.To establish a rat model of chronic PAH that similar to human beings.2.To observe the expression of CPT1 in the rats with PAH and to explore its role in pulmonary vascular remodeling.3.To investigate the role of CPT1 in the proliferation and migration of PASMCs and its signal transduction mechanism.Methods1.Novel PAH rat model130 male Sprague-Dawley rats were randomly divided into three groups,including normal control,conventional,and novel.In conventional,a single dose of 40 mg/kg MCT was given intraperitoneally to induce PAH.Animals in the novel received twice intraperitoneal injection of 20mg/kg MCT in a week.Mean pulmonary arterial pressure?mPAP?,right ventricular hypertrophy index?RVHI?and pulmonary vascular remodeling?the percentage of total wall thickness to external diameters of pulmonary arterioles diameter[WT%]and the percentage of wall area to the total area of vessels[WA%]?were assessed at the week 1,2,3 and 4 after first MCT administration.The numbers of survival rats were recorded till week 4.2.In vivo experimentsA rat model of PAH was established by intraperitoneal injection of MCT 20mg/kg fortnightly.2 and 4 weeks after the first injection of MCT,the relevant indicators of lung tissue were collected.The expression and localization of CPT1 in lung tissue were observed by immunofluorescence confocal microscopy with?-smooth muscle actin??-SMA?and CPT1 monoclonal antibody.Western blot was used to detect the expression of CPT1,p-AMPK/AMPK,PCNA,p53 and p21 in lung tissue.Non-radiative method was applied to determine CPT1 activity,and the chemiluminescence method was used to detect ATP production.3.In vitro experimentsPASMCs were isolated from the male Sprague-Dawley rats.Smooth muscles specific markers?-SMA,CPT1 monoclonal antibody immunofluorescence staining were used to identify PASMCs and determine the expression and localization of CPT1in PASMCs.After 24hs of incubation,treatment of PASMCs with PDGF?platelet-derived growth factor?-BB and CPT1 irreversible inhibitor Etomoxir was carried out.The CPT1a gene was constructed into lentiviral vector to overexpress CPT1in PASMCs.The proliferation of PASMCs was detected by MTT assay.The expression of CPT1,p-AMPK/AMPK,p53 and p21 was detected by Western blot.Non-radiative method was applied to determine CPT1 activity,and the chemiluminescence method was used to detect ATP production.Cell cycle was measured by flow cytometry.4.Statistical AnalysisThe data were expressed as the mean±standard deviation.Student t-test,one-way and two-way analysis of variance?ANOVA?were used to test the differences,followed by SNK-q test for pairwise comparisons.Survival curves were derived using the Kaplan-Meier method and compared using a log-rank test.The Statistical Package for Social Sciences?SPSS,version 13.0,Chicago,IL?was used for all statistical analyses.P<0.05 was considered significant.Results1.Novel PAH rat model4 weeks after first MCT injection,mPAP?conventional 34.48±2.36mmHg,novel31.58±2.69mmHg vs.control 17.30±3.10mmHg;P<0.05?,RVHI?conventional53.68±5.70%,novel 45.07±11.21%vs.control 25.73±6.04%;P<0.05?,WA%?conventional 80.57±3.92,novel 80.49±7.63 vs.control 47.03±6.95;P<0.05?and WT%?conventional 58.50±3.27,novel 56.33±6.87 vs.control 28.21±3.94;P<0.05?in conventional and novel were higher than control.Compared with conventional,mPAP and RVHI were lower in novel?P<0.05?.On the 4^th week 4 after the first MCT injection,survival percentage in novel was higher than that in conventional?78.57 vs.57.14%;P=0.027,n=22?.2.In vivo experimentsCPT1 was expressed in rat lung tissue.The expression of CPT1 in pulmonary arteries of normal rats was lower than that in rats after 2 and 4 weeks of MCT injection.But the expression level of CPT1 in pulmonary arteries in rats after 2 weeks of MCT injection was higher than 4 weeks.2 and 4 weeks after MCT injection,pulmonary artery smooth muscle layer in PAH rats was significantly thickened and pulmonary vascular structure remodeled.The activity of CPT1 and ATP production were increased in the lung tissue of PAH,but the difference was not significant at 2 and 4 weeks.The phosphorylation of AMPK and the expression of p53 and p21 were decreased during the progression of PAH.The phospho-AMPK and p53 were significantly decreased from 2weeks,while p21 was obviously declined at 4 weeks.In addition,PDGF and PCNA were significantly increased.3.In vivo experimentsAfter successful isolation and culture of SD rat's PASMCs,?-SMA immunofluorescent staining confirmed that isolated cells were smooth muscle cells.Immunocytofluorescence staining showed that CPT1 could express in PASMCs and locate in mitochondria.PDGF-BB upregulated the expression of CPT1 in a concentration and time-dependent manners and promoted the proliferation of PASMCs.PDGF-BB could promote the formation of ATP,inhibit phosphorylation of AMPK,decrease the expression of p53 and p21,reduce the ratio of G0/G1 phase in the cell cycle and increase the ratio of G2/M phase,which can be reversed by CPT1 inhibitor Etomoxir.Overexpression of CPT1 can promote the proliferation of PASMCs and ATP production,and inhibit the phosphorylation of AMPK,p53 as well as p21.In PASMCs,CPT1 could promote the cell migration ability.Conclusions1.Novel use of twice intraperitoneal injection of 20mg/kg MCT could induce chronic PAH rat model successfully and increase survival percentage compared with single injection of 40mg/kg MCT in rats.2.The expression of CPT1 in PASMCs is involved in the formation of PAH.The mechanism may include the abnormal proliferation and migration ability of pulmonary artery smooth muscle cells.3.CPT1 may be one of the regulatory targets for the proliferation and migration of PASMCs.CPT1 may regulate cell proliferation through AMPK-p53-p21 pathway.