The Role And Underlying Mechanism Of Cyclin-dependent Kinase 9 (CDK9) In Pulmonary Hypertension

Background: Pulmonary artery hypertension(PAH)is a serious type of cardiopulmonary vascular disease with high risk of clinical anesthesia.Pulmonary vascular remodeling and excessive proliferation of pulmonary artery smooth muscle constitute an important pathological feature of pulmonary hypertension.There are some similarity between the pathology of pulmonary artery smooth muscle hyperproliferation and tumor,cyclindependent kinase 9(CDK9)is currently an important anti-tumor target,but the role of CDK9 in pulmonary hypertension is still unclear.Objective: To investigate the expression of CDK9 in pulmonary artery smooth muscle and the effect of CDK9 inhibitor on pulmonary vascular remodeling and pulmonary artery smooth muscle hyperproliferation in pulmonary hypertension and its potential molecular mechanism.Methods: The expression of CDK9 in pulmonary arterial smooth muscle of pulmonary hypertension was studied by in vivo pulmonary hypertension model and in vitro culture of human pulmonary vascular smooth muscle cells(HPASMC)hypoxia model.In the in vivo experiment,adult male Sprague-Dawley rats were injected intraperitoneally with monocrotaline(MCT,60 mg/kg,ip)to successfully prepare pulmonary hypertension model.The pulmonary arteries of normal control group and MCT group were dissected,and the expression of CDK9 m RNA and protein in pulmonary artery smooth muscle was detected by RT-PCR and Western-Blot,respectively.The in vitro model was treated with human pulmonary artery smooth muscle cells(HPASMC),and 3%O2,5% CO2 was treated with hypoxia for 0,1,3,6,12,24 h,respectively.The protein expression of CDK9 in HPASMC was detected.Secondly,HPASMC detected changes in CDK9 m RNA levels after 24 h of hypoxia.The inhibitory effects on pulmonary vascular remodeling and hyperproliferation of pulmonary artery smooth muscle by treatment of CDK9 inhibitor Flavopiridol(Fla).SD rats were randomly divided into three groups: Control group,MCT group,MCT + Fla group. Successfully modeled 21 days after intraperitoneal injection of MCT in rats.The CDK9 inhibitor Flavopiridol(5 mg/kg,ip)was started 7 days after MCT injection,twice a week for 14 days.The right ventricular systolic pressure was measured after the end of the experiment.The pulmonary arterioles and heart cross sections were observed by HE staining.The percentage of wall thickness(WT%)and the percentage of wall area(WA%)were calculated,weighed and then calculated the right ventricular hypertrophy index(RV/LV+S).Immunofluorescence was used to detect the expression of α-SMA and PCNA in pulmonary arterioles.In vitro,HPASMC were treated with different concentrations(0.025,0.05,0.1,0.5,1 μM)of Flavopiridol,followed by hypoxia treatment for 24 h in 3% O2,5% CO2,and cell viability was measured by CCK8.Flavopiridol was selected at a concentration of 0.1 and 0.5 μM for subsequent experiments.The experimental groups were: Control,Hypoxia,Hyx+Fla,and the Ed U kit was used to detect cell proliferation,and the apoptosis rate was detected by flow cytometry.The specific mechanism of CDK9 promoting the proliferation of pulmonary artery smooth muscle cells was studied.The pulmonary artery of control group,MCT and MCT+Fla were dissected.The phosphorylation level of RNA pol Ⅱ CTD-Ser2 locus was detected by WB.The m RNA and protein expression levels of downstream c-Myc,Mcl-1 and Survivin were detected by RT-PCR and WB,respectively.The m RNA expression levels of negative regulators of CDK9,Larp7,HEXIM1 and Mepc E were detected by RTPCR.Results: In the rat pulmonary hypertension model,the mRNA and protein expression of CDK9 in pulmonary artery of MCT group was increased compared with Control group(P<0.01).In vitro hypoxia model,the expression of CDK9 protein in HPASMC was timedependent,CDK9 protein expression was down-regulated in hypoxia at 1-6 h,and then increased continuously,the most significant increase was observed at 24 h(P<0.05),and CDK9 m RNA was up-regulated 24 h after hypoxia(P<0.05).By compared with the Control group,the right ventricular systolic pressure(P<0.001)and right ventricular hypertrophy index(P<0.01)were increased significantly in MCT group.HE staining showed significant thickening of the pulmonary arteriolar wall,both WA% and WA% increased significantly(P < 0.001).After using the CDK9 inhibitor Flavoripidol,compared with the MCT group,the right ventricular systolic pressure(P<0.01)and the right ventricular hypertrophy index (P<0.01)were significantly decreased,the pulmonary arteriolar wall thickening was reduced,and both WT% and WA% were decreased(P < 0.05),pulmonary hypertension was partially relieved.After 24 hours of hypoxia,the cell viability of HPASMC increased(P<0.01),the proportion of proliferating cells increased(P<0.01),and the apoptosis rate decreased(P<0.05).The use of Flavopiridol inhibited cell proliferation in a concentration-dependent manner(P<0.01),decreased the proportion of proliferating cells(P<0.01),and increased the apoptotic rate(P<0.01).In addition,compared with the Control group,the phosphorylation level of the RNA pol Ⅱ CTD-Ser2 locus in the pulmonary arteries of the MCT group was increased,and the expression of c-Myc,Mcl-1 and Survivin m RNA and protein were increased.The expression levels of Larp7,HEXIM1 and Mepc E m RNA were decreased.The phosphorylation level of the RNA pol Ⅱ CTD-Ser2 locus in the MCT+Fla group was decreased,and the expression levels of c-Myc,Mcl-1 and Survivin m RNA and protein were decreased.Conclusion: In pulmonary hypertension,CDK9 expression is up-regulated in pulmonary artery smooth muscle.CDK9 inhibitor can significantly inhibit excessive proliferation of pulmonary artery smooth muscle cells and relieve pulmonary hypertension.Increased CDK9 can increase the phosphorylation of Ser2 residues in the CTD region of RNA pol Ⅱ,thereby enhancing the transcriptional elongation process,increasing the transcription and protein expression levels of the downstream pro-proliferative gene c-Myc and anti-apoptotic genes Mcl-1 and Survivin,triggering pulmonary artery smooth muscle cells proliferate excessively.Furthermore,in pulmonary hypertension,downregulation of the negative regulatory related molecules Larp7,HEXIM1 and Mepc E may be involved in the increased activity of CDK9.