The Mechanism Of FoxM1 Regulating DNA Damage Response Of Pulmonary Artery Smooth Muscle Cells In Mice With Pulmonary Arterial Hypertension Via NBS1

Objective:Pulmonary arterial hypertension(PAH)is a multifactorial pulmonary vascular disease with elevated pulmonary artery pressure.The pathological feature is pulmonary vascular remodeling(PVR),and its pathogenesis is not fully understood.The available drugs for PAH therapy can’t completely reverse the established PVR,which is the main reason for the poor prognosis and low long-term survival rate of patients with severe PAH.Therefore,clarifying the pathogenesis of PVR and finding new theraputic targets for PAH have important guiding significance for the clinical treatment dilemma faced by the disease.The latest researches in this field show that severe PAH has "tumor-like" characteristics in pathology.Through comparative screening,we select Forkhead box protein M1(FoxM1),a multifunctional transcription factor that plays an important role in the occurrence and development of tumors,to explore its role and mechanism in PAH.In this study,we first investigate the differences and characteristics of PVR in PAH mouse models induced by left pneumonectomy,jugular injection of monocrotaline pyrrole and left pneumonectomy combined with jugular injection of monocrotaline pyrrole,respectively,and then investigate the expression of FoxM1 in lung tissues of PAH mouse model and its regulatory effect on DNA damage response of pulmonary artery smooth muscle cells(PASMCs).Furthermore the effect and mechanism of FoxM1 on the cell proliferation,migration and cell cycle distribution of PASMCs are analyzed at the cellular level.Materials and Methods:1.Three mouse modles of PAH were established induced by left pneumonectomy(PE group),jugular injection of monocrotaline pyrrole(MCTP group)and left pneumonectomy combined with jugular injection of monocrotaline pyrrole(P+M group),respectively.Hemodynamic and morphological parameters were performed under the same condition to compare the differences and characteristics of PVR in above-mentioned three models,including right ventricular systolic pressure(RVSP)、right ventricle/(left ventricle plus septum)(RV/LV + S),percent of wall thickness in the pulmonary artery(WT %),muscularization of non-muscular arteries,neointimal formation and vascular obstruction score(VOS).Systemic noninvasive plethysmography was used to detect whether there were differences in lung function.2.A mouse PAH model was established induced by left pneumonectomy combined with jugular injection of MCTP,and then FoxM1 was inhibited by continuous intraperitoneal injection of thiostrepton,a kind of FoxM1 inhibitor.Clinical manifestations,hemodynamic indexes and histopathological changes were observed in each group to evaluate the effect of FoxM1 inhibitor on PVR in PAH model mice.Immunohistochemistry staining,real-time fluorescent quantitative PCR(RT-qPCR)and Westertn blot were used to detect the mRNA and protein levels of HIF-1a,FoxM1,NBS1,ATM and key genes in the ATM pathway in the lung tissues of each group,and to explore the regulatory effect of FoxM1 on DNA damage response in PAH.Transcriptome sequencing was performed on the lung tissues of mice in control group,PAH model group and FoxM1 inhibitor intervention group to screen out the important molecules and signaling pathways related to FoxM1.3.PASMCs of normal mice were obtained by type I collagenase digestion method,and then transferred to the third generation of culture.PASMCs were identified by a-smooth muscle actin immunofluorescence staining,and cells with a positive rate of ≥95% were used for subsequent experiments.In vitro,different concentrations of recombinant mouse PDGF-BB and thiostrepton were used to treat PASMCs.MTT assay,flow cytometry and cell scratch test were used to detect the effects of PDGF-BB and thiostrepton on the cell proliferation ability,cell cycle and migration of PASMCs,respectively.The expressions of FoxM1,NBS1 and ATM in PASMCs were detected by RT-qPCR and Western blot to explore the regulation of FoxM1 on DNA damage response.Results:1.Compared with the control group,mice in PE group,MCTP group and P+M group did not show shortness of breath,hair standing or depilation during the whole experiment.However,the mice weight decreased significantly in MCTP group and P+M group.Model mice of the three groups were tested for hemodynamic indexes and histopathological changes on the 21 st,35th,and 49 th days after pneumonectomy,respectively.The results showed that there were no statistically significant differences in RVSP,RV/LV+S,WT%,and muscularization of non-muscular arteries between PE group and control group,and there was no neointima formation in PE group.In MCTP group,RVSP increased from the 21 st day after pneumonectomy,and reached the peak on the 35 th day,with slight right ventricle hypertrophy,pulomonary arteries thickness,and non-muscular pulmonary arteries muscularization,but neointima formation and plexiform lesions were not observed.In the P+M group,hemodynamic and histopathological changes were similar to those in the MCTP group,but more significant.What’s more,neointima formation and suspected plexiform lesion in the acinus pulmonary arteries were observed,leading to obvious lumen stenosis.Compared with the control group,P+M group,MCTP group and PE group showed no difference in lung function.2.The levels of FoxM1 mRNA and protein in lung tissues of mice in PAH model group were significantly higher than those in control group.After mice with PAH were treated with thiostrepton,the body weight gain recovered,hemodynamic parameters and pathological indexes reflecting the degree of PVR were attenuated,and there was no neointima formation.The levels of HIF-1a,NBS1,ATM,Chk2 and P21 mRNA and protein were all highly increased in PAH model mice detecting by IHC staining,RT-qPCR and Western blot.The level of P53 mRNA was decreased,but protein content was increased.With the inhibition of FoxM1 expression,the levels of NBS1,ATM,and Chk2 also decreased,but the expressions of HIF-1a,P53 and P21 were not changed.The protein contents of NBS1 had a linear positive relationship with the contents of FoxM1(r=0.617,P=0.033).The transcriptome sequencing showed that both GO functional classification and KEGG pathway analysis of the top differential genes suggested predominant immunoinflammation,in which the Prkd3-Rap1 pathway was lowly expressed in the PAH model group,while the expression was increased in the thiostrepton intervention group.3.The experssions of FoxM1,NBS1 and ATM in PASMCs treated with PDGFBB were significantly higher than those in control group,and the cell proliferative potential and migrtion were enhanced,and the ratio of G2-M phase in the cell cycle was significantly increased.As the expression of FoxM1 in PASMCs was inhibited by thiostrepton,the expressions of NBS1 and ATM decreased,accompanied by the weakening of cell prolifertation and migration ability and cell cycle arrest in the G0-G1 phase.Conclusion:1.Left pneumonectomy could not induce the establishment of PAH mouse model.Jugular injection of MCTP could induce mild PAH without neointima formation.Left pneumonectomy combined with jugular intravenous injection of MCTP could induce severe PAH formation with the pathological hallmark: neointima formation.2.FoxM1 could promote PAH formation by regulating the ATM-Chk2 pathway to participate in DNA damage response via NBS1,contributing to cell cycle arrest,DNA damage repair and cell survival.3.Both HIF-1α and P53-P21 pathway were activated in PAH,but there expressions were not affected by FoxM1.4.Immunoinflammation played an important role in the pathogenesis of PAH,but whether it was regulated by FoxM1 and the relationship between Prkd3-Rap1 signaling pathway and FoxM1 remained to be further explored.5.Recombinant PDGF-BB could upregulate FoxM1 in PASMCs of normal mice,and promote cell cycle progression,cell proliferation and migration by participating in DNA damage response through the positive regulation of FoxM1 on NBS1 and ATM.