The Function And Mechanisms Of Structural Maintenance Of Chromosomes 4 In Pulmonary Vascular Remodeling

BackgroundPulmonary arterial hypertension(PAH)is a devastating syndrome typified by the hyperproliferation of pulmonary vascular cells,mainly pulmonary artery smooth muscle cells and endothelial cells.The excessive cell growth contributes to progressive obstruction of the pulmonary vasculature,which eventually produces increased pressure in the lungs and right ventricle,resulting in irreversible damage.The pathogenesis of PAH is not completely clarified,but pulmonary vascular remodeling(PVR)plays a core function in the progression of PAH.No specific cure is currently available since current treatments for PAH mainly concentrate on the vasoconstriction mechanisms and incomplete understanding of vascular remodeling.Therefore,it is crucial to improve the understanding of the cellular and molecular mechanisms in the development of PVR and to find new effective targets for the treatment of PVR.Researches have confirmed that aberrant hyperproliferation,migration and resistance to apoptosis of pulmonary artery smooth muscle cells(PASMCs)are the primary pathologic and physiologic underpinnings of PVR.Structural maintenance of chromosomes 4(SMC4)is one of the subunits of the chromosome structural maintenance protein family,which forms a condensin with SMC2 during mitosis and acts on chromosome cohesion and segregation.Recent studies have illustrated that SMC4 was associated with a variety of non-mitotic cellular functions,including the processes of heterochromatin organization,maintenance of a silent state of gene expression,and DNA repair.Increasing evidence have demonstrated that SMC4 is highly associated with tumorigenesis and is involved in regulating altered proliferation and migration functions of various cancer cells.Previous studies have demonstrated the involvement of multiple tumor mechanisms in PAH pathogenesis and the presence of a large number of smooth muscle cells with carcinoid proliferation in the remodeling vascular zone.Previous bioinformatics studies have revealed significant differences in SMC4 expression in lung tissue from PAH patients and healthy individuals,and analysis of the lung development dataset GSE43767 expression network showed that SMC4 expression decreases with lung developmental maturation and increases within proliferatively active lung adenocarcinomas.Recent studies have shown that SMC4 expression levels significantly correlate with the cell cycle and are involved in the regulation of cell differentiation,proliferation and apoptotic processes.Therefore,we hypothesized that SMC4 is highly expressed in the PVR and is involved in the pathological proliferation of PASMCs and PVR development,but the role of SMC4 in the lung tissue of monocrotaline(MCT)-induced PVR rats and its underlying mechanisms still unclear.Objectives1.To determine the expression of SMC4 in the pulmonary artery tissue and PASMCs of PVR rats.2.To investigate the effect of SMC4 on the cellular function of PDGF-BB-induced PASMCs.3.To investigate the potential mechanism of the effect of SMC4 on PVR.4.To provide novel potential therapeutic target for PVR and PAH treatment.Methods1.Animal experiment:SD rats were randomly divided into 4 groups:control,pulmonary vascular remodeling group(MCT),intervention control group(MCT+Lenti-NC),and experimental intervention group(MCT+Lenti-SMC4).The expression of SMC4 in rats was interfered by administering lentivirus intranasal instillation for 2 weeks,lung tissues were collected and examined for the effect of SMC4 knockdown,while a pulmonary vascular remodeling model was developed by a single intraperitoneal injection of monocrotaline(60 mg/kg)for 4 weeks.Lung tissues were stained with H&E to observe the morphological and structural changes of arteries and vascular remodeling.Immunofluorescent double staining was performed to identify the expression and localization of SMC4 and α-smooth muscle actin(αSMA).2.Cell experiment:Rat pulmonary artery smooth muscle cells were extracted and classified into 4 groups:Control,PDGF-BB,Si-NC and Si-SMC4.QRT-PCR and Western Blotting were applied to assess SMC4 expression;cell proliferation was detected by CCK8 and EDU.Western Blotting was conducted to identify the expression of apoptotic proteins;wound healing and transwell assays were conducted to determine the cell migration;Western Blotting assayed the phenotypic switch and NF-κB pathway-related proteins.Results1.The expression of SMC4 is increased in MCT-induced rat pulmonary artery tissueThe results of qRT-PCR and Western Blotting revealed a dramatic increase of SMC4 in the MCT group;immunofluorescence staining of lung tissues confirmed a notable increase in SMC4 in the remodeled pulmonary artery vessels.2.The expression of SMC4 is elevated in PDGF-BB-induced rat pulmonary artery smooth muscle cellsWe incubated PASMCs respectively with 0,1,10 and 20 ng/ml concentrations of PDGFBB for 24 h.QRT-PCR and Western Blotting discovered that SMC4 levels were seriously higher relative to the control group under stimulation conditions with 20 ng/ml drug concentrations,both as regard to RNA level and protein levels.3.SMC4 facilitates PDGF-BB-induced proliferation,migration and anti-apoptotic capacity of rat pulmonary artery smooth muscle cellsWe used small interfering RNA targeting SMC4 to assess whether SMC4 regulates PDGFBB-induced proliferation and migration of PASMCs.CCK8 and EDU results proved that knockdown of SMC4 hindered PDGF-BB-induced cell proliferation,while Western Blotting results indicated that knockdown of SMC4 enhanced PDGF-BB-inhibited Bax and cleaved caspase 3 expression,inhibited PDGF-BB-induced Bcl-2 expression,and accelerated PASMCs apoptosis inhibited by PDGF-BB.In the distal portion of the pulmonary vascular remodeling,PASMCs migrate from the medial layer to the lumen.We estimated whether SMC4 is indispensable for PDGF-BB-induced PASMC migration.The outcomes of trans well and wound healing assays exhibited that SMC4 interference apparently impaored the migration of PASMCs.4.SMC4 advances PDGF-BB-induced phenotypic switch of rat pulmonary artery smooth muscle cellsWe exploited small interfering RNAs against SMC4 in PASMCs.Western Blotting showed that knockdown of SMC4 markedly inhibited PDGF-BB-induced expression of Collagen-1 and OPN,while increasing the expression of the inhibited α-SMa and SM22a,suggesting SMC4 advanced PDGF-BB-induced phenotypic switch of PASMCs.5.SMC4 may contribute to alterations in PASMCs function through activation of NF-κB signaling pathwayWe addressed PASMCs with the NF-κB pathway inhibitor BAY11-7082 and small interfering RNA against SMC4,separately,and discovered that knockdown of SMC4 prominently restrained the level of PDGF-BB-induced phosphorylation levels of the NF-κB pathway.This effect was consistent with the effect of BAY 11-7082.6.Knockdown of SMC4 abrogates pulmonary vascular remodeling and NF-κB phosphorylation in MCT rats.We used a lentivirus targeting SMC4 to knock down SMC4 in rats,and Western Blotting revealed that knocking down SMC4 significantly abolished pulmonary vascular remodeling and phosphorylation of the NF-κB pathway in MCT rats.Conclusions1.SMC4 is substantially increased in MCT-induced PVR rats and PDGF-BB-induced PASMCs.2.SMC4 advances PDGF-BB-induced proliferation,migration and apoptotic resistance in PASMCs.3.SMC4 propels PDGF-BB-induced phenotypic switch of PASMCs.4.SMC4 may be involved in PVR development through NF-κB pathway.