The Effects And Mechanisms Of Overexpression Of Endothelin-1 On The Apoptosis And Hypertrophy Of Rat Pulmonary Arterial Microvascular Smooth Muscle Cells In Vitro

IntroductionPulmonary arterial hypertension(PAH),defined as a mean pulmonary artery pressure(mPAP) greater than 25 mmHg at rest or 30 mmHg with exercise and a pulmonary capillary wedge pressure less than 15 mmHg,as measured by right heart catheterization,is a multifactorial,serious and often progressive disorder that results in a sustained increase in pulmonary vascular resistance,right ventricular dysfunction and impairment in activity tolerance,and may lead to right-heart failure and death. The pathogenesis of PAH is complex and incompletely understood,but may share common histological abnormalities and pathophysiology.The irreversible morphologic changes in the pulmonary vascular bed lead to vascular remodeling including distal neomuscularization of the arterioles,intimal thickening,and medial hypertrophy.Abnormal collagen matrix is deposited within the adventitia later.Many of these changes can be attributed to the broken of the balance between hyperplasia/ hypertrophy and apoptosis of pulmonary arterial microvascular smooth muscle cells (SMC) and attention has therefore been focused on the related regulatory mechanisms of vascular SMC involved.Therefore,the successful isolating and culturing of the pulmonary arterial microvascular SMC in vitro represents the important model in the exploring of the pathogenesis of PAH.Endothelin-1(ET-1) is a potent 21 amino-acid peptide and mainly generated from the endothelium of blood vessels,as well as vascular SMC in part,and functions as a vasodilator and vasoconstrictor in a paracrine and autocrine manner on the ETA and ET_B receptors,which has been suggested to contribute to the local homeostasis of the pulmonary vasculature.Importantly,the amounts of ET-1 released by vascular SMC under inflammatory conditions are equivalent to those produced by the endothelium.Further,as the number of the vascular SMC in remodeling vessels is potentially greater than that of endothelial cells,the endogenous ET-1 production by these cells becomes increasingly important.In PAH patients,ET-1 is abnormally up-regulated in the circulation and pulmonary arteries,which is strongly correlated with measures of disease severity and survival in patients.Therefore,besides its vasoaction effects,ET-1 has been suggested to possess a direct mitogenic action, and/or apoptosis inhibition effect of vascular SMC in PAH.To investigate the effect of the hyperplasia,hypertrophy and apoptosis properties of the pulmonary arterial microvascular smooth muscle cell under the stimulating of the increasing ET-1 will pave a way to understand the pathophysiology of PAH,especially inducing some new point in preventing and treating strategies.In our current study,based on the successful culturing of rat pulmonary arterial microvascular smooth muscle cells(RPMC),we investigated the effects of ET-1 overexpression on the hyperplasia,hypertrophy and apoptosis characteristics of RPMC and the possible signaling pathway involved was also explored through transiently transfecting RPMC with ET-1 gene.Partâ… Method to cultivate rat pulmonary arterial microvascular and large arterial smooth muscle cells and comparison of their proliferative activityObjective:To define the method to cultivate RPMC and rat large arterial smooth muscle cells(RPLC) and to compare their proliferative activity.Methods:The pulmonary artery of an anesthetized adult rat was cannulated via the right ventricle.After orderly perfused with PBS,trypsin,low melting-point agarose and iron oxide particles mixture,the left atrium and pulmonary artery was clamped. Then the trachea was cannulated,and lung air space was filled with low melting-point argarose and the trachea was clamped.The whole lung and heart were isolated and transferred into the culture mediumâ… [Dulbecco's modified Eagle's medium with penicillin(100 U/ml) and streptomycin(100μg/ml)]on ice to set the agarose.After subsequent trimming of the subpleural margin(no more than 1mm),the lung was sliced and inspected with the microscope to ensure vessels of size less than 200μm. Following digested with collagenaseâ…£,rinsed with the culture mediumâ… ,the residue tissues were minced and suspended with culture mediumâ…¡[20%fetal bovine serum (FBS) in the culture mediumâ… ]and then resuspended into the 6-well culture plate and incubated in humidified air with 5%CO₂ at 37℃.Meanwhile,same as the procedure, the first and second conduct artery were isolated after the trypsin perfusion finished with the microscope and dipped into collagenase to digest the adventitia.The aorta was clipped and the adventitia,the intima and the outer portion of the media of artery were mechanically scraped with the forceps.After repeated rinsed,the residuum was cut into approximately 5×5mm squares in culture plate and placed in an incubator under the same culture circumstance.The RPMC and RPLC were identified by immunofluorescence staining and electron microscope technology.Cell proliferation viability was tested by MTT assay.Flow cytometry was used to assess the cell cycle.Result:From 3 to 5 days,cells were visible migrating from the explants in RPMC, while from 7 to 10 days,cells were visible migrating from the explants in RPLC and at confluence,and both of the cells were elongated in shape and formed the typical "hill and valley" formation of cultured smooth muscle cells under the inverted contrast microscope.The cells were identified with the characteristicα-actin by the immunofluorescence and great amount of Intermediate filaments,dense plaques and dense bodies by electron microscope.The purity of the 3rd passage RPMC and RPLC was more than 95%.Compared with RPLC,RPMC exhibited a faster growth rate in 4d with MTT and higher cell Proliferate Index with Flow cytometry.Conclusion:The method to cultivate RPMC via tissue perfusion is feasible and there is a different proliferative characteristic between RPMC and RPLC.Further researches using this cell model may pave a way to explore the pathogenesis of the lung diseases with pulmonary arterial microvascular smooth muscle cell disorders,e.g. pulmonary hypertension. Partâ…¡The effect and mechanism of overexpression of endothelin-1 on apoptosis of rat pulmonary arterial microvascular smooth muscle cells in vitroObjective:To investigate the effect and mechanism of overexpression of ET-1 on the apoptosis of RPMC in vitro.Methods:The RPMC was obtained and cultured by the lung tissue perfusion method, then the third generation RPMC was transient transfected with the pMEXneo-ET1 and pCDNA5-FRT-TO-ET1-3'UTR plasmids as well as the empty vector respectively via Lipofectamine^TM 2000.Real time RT-PCR was used to assess the ET_A and ET_B levels and flow cytometry was used to assess the apoptosis of RPMC.Akt and Caspase-3 were detected by Western blot assay.Results:The transfection efficiency is about 25%.The mRNA of ET_A receptor was higher than that of ET_B receptor.Both of the ET_A receptor and the ET_B receptor mRNA in the transfected RPMC was increased,however,the ratio between the ET_A receptor and the ET_B receptor mRNA was the same as the control.Flow cytometry analysis revealed that the apoptosis was decreased after RPMC was transfected with the ET-1 for 48 h.The Western blotting results showed that overepression of ET-1 in RPMC increased the phosphorylation of Akt and reduced the Cleaved Caspase-3.Conclusions:Overexpression of the ET-1 inhibits the apoptosis of RPMC and activation of Akt/PKB-Caspase-3 signaling pathway may be involved in its mechanism,which may play a role in the remodeling of the pulmonary microvascular arteries.Partâ…¢The effect and mechanism of overexpression of endothelin-1 on hypertrophy of rat pulmonary arterial microvascular smooth muscle cells in vitroObjective:To investigate the effects and mechanism of overexpression of ET-1 on the hypertrophy of RPMC in vitro.Methods:Lung tissue perfusion method was used to obtain the primary RPMC and it was identified via immunofluorescence staining and electron microscope technique. The RPMC was transient transfected with the pMEXneo-ET1 and pCDNA5-FRT-TO-ET1-3'UTR plasmids as well as the empty vector respectively via Lipofectamine^TM 2000.Flow cytometry was used to assess the cell cycle and cell size of RPMC,as well as the ratio of the protein/DNA of the transfected RPMC on the 72 h.Akt and mTOR were detected by Western blot assay to investigate the molecular mechanism.Results:Primary RPMC was obtained successfully,and the mRNA of ET-1 was higher than that of the empty vectors.Flow cytometry analysis revealed that the cell size was increased after RPMC was transfected with the ET-1 for 72 h.The ratios of the protein/DNA of the transfected RPMC were extended than the empty vectors on 72 h.The Western blotting results showed that overepression of ET-1 in RPMC increased the phosphorylation of Akt and mTOR.Conclusions:Overexpression of the ET-1 induces the hypertrophy of RPMC and activation of Akt/PKB-mTOR signaling pathway may be involved in its mechanism, which may be responsible for the remodeling of the pulmonary microvascular arteries.