Effect Of Mfn2on Hypoxia Pulmonary Vascular Smooth Musle Cells And Its Underlying Mechanism

Background:Pulmonary arterial hypertension (PAH) is a circulatory diseases which is characterized by vascular wall excessive proliferation,eventually leading to the pulmonary artery lumen obstruction.Hypoxia pulmonary hypertension (HPH) is one of the most common types of pulmonary arterial hypertension(PAH). The pathophysiological process of HPH include hypoxia pulmonary vasoconstriction(HPV) and hypoxic pulmonary vascular remodeling(HPVR).And PASMCs (pulmonary arterial smooth muscle cells) excessive proliferation is the most important pathological mechanism of HPVR. Mfn2is not only an important element in maintaining the mitochondria normal morphology,but also an important intracellular signal transduction molecule which particular play an important role in cell proliferation and apoptosis pathway. Studies have reported that over-expression of Mfn2in vascular smooth muscle cells (VSMCs) could inhibit the PI3K/Akt pathway, increase permeability of the outer mitochondrial membrane, release cytochrome C from mitochondria to cytoplasm and then activate downstream caspase cascade、at last promote VSMCs apoptosis. In cardiovascular diseases, the effect of Mfn2in the proliferation of VSMCs has been researched much more in-depth, but little studied in pulmonary hypertension. In view of that Mfn2play an important role in VSCMs proliferation, we speculate that Mfn2play the same effect in the PASMCs of HPH.Objectives:In this study, we will study the expression of Mfn2in the rats lung tissue of HPH desease model and in the PASMCs during hypoxia. And we will further investigate the effect and mechanism of Mfn2in the balance of PASMCs proliferation and apoptosis which may have an effect in the PI3K/Akt signal pathway and mitochondria apoptosis pathway.Methods:Part I and vivo animal studies:(1)we divided the20SD rats into normoxic and hypoxic group randomly and equally. The rats in hypoxic group were feeding daily in continuous hypoxia circumstance at the oxygen concentration of (10±0.5)%in home-made plexiglass box(0.8m×0.55m×0.40m) for4weeks,8hours a day.The rats in normoxic group were feeding daily in normoxia oxygen concentration (21%) and reared under the same living conditions as hypoxic group;(2) After the intervention finished, all the rats were measured by lung function、mean pulmonary artery pressure(mPAP), right ventricular hypertrophy index(RVHI) to identify the hypoxia induced HPH model;(3) RT-PCR and Western blot(WB) to detect Mfn2mRNA and protein expression in normoxic and hypoxia-induced HPH model of rats lung tissue.And the expression of PCNA protein was detected. Part II and vitro study:(1) We referenced the patch-attaching method for primary culture of rat’s PASMCs.And PASMCs a-actin was characterized by immunofluorescence;(2)In order to verify PASMCs proliferation of time-dependent and filter out the best oxygen concentration for PASMCs proliferation, PASMCs were cultured under different time periods in hypoxia incubator(oxygen concentration2.5%). Normoxic and hypoxic group were cultured in hypoxia incubator for6h,12h,24h,48h respectively at the same time.We applied MTT assay to test the cells proliferation and used RT-PCR, WB technology to detect the PASMCs Mfn2mRNA and protein expression、PCNA protein expression after hypoxia for Oh、6h、12h、24h、48h;(4) After the former experiment have filtered out the optimal time to hypoxia intervention, we would choose the time for the latter study. We simultaneously or respectively adopted LY294002(PI3K inhibitor) and overexpression Mfn2by plasmid which is carried pEGFPMfn2and transfected by LipofectamineTM2000. After the intervention finished, We verified the transfection efficiency by detecting Mfn2protein expression.This part we divided the PASMCs into6groups and named like this:normoxia、hypoxia group、hypoxia+shNC (transfected with empty plasmid group)、hypoxia+shfn2(overexpression Mfn2group), hypoxia+LY294002and hypoxia+LY294002+shMfn2.After these intervention finished,we applied the MTT assay and flow cytometry to test the PASMCs proliferation and apoptosis respectively.Then used WB technology to test the Mfn2p-Akt (phosphorylation type of Akt)、mitochondrial cytochrome C、cleaved caspase9protein expression and we tested β-actin as internal protein.Results:In vivo animal research:(1)Mfn2mRNA and protien expresssion were reduced and PCNA protein expression increased in the HPH model of rats’s lung tissue. In vitro cell research:(1) MTT assay resulted that from hypoxia for6h PASMCs cell number were beginning to increase, and the increased trend was reached to the highest after hypoxia for24h. Compared with normoxic group cell number,hypoxia for6h、12h、24h、48h were increased and the increased was statistically significant. Compared with hypoxia for12h, the24h and48h cell number were increased and the increased was statistically significant.Compared with hypoxia for24h,the48h cell number although increased but the difference was not statistically significant.The PASMCs Mfn2protein expression began to decline from6h and the variation trend was the same as the MTT assay results. PCNA protein expression began to increase from hypoxia for6h, and the variation trends was the same as the MTT assay results too.(2) After by WB technology detecting Mfn2mRNA and protein expression, we found that in over-expression group Mfn2protein expression was increased compared to the non-transfected and transfected with the no-targets gene group. And the increase was statistically significant.There was no statistically significant change between non-transfected and no-targets gene.(3)By the above results, we filtered out the optimal time for hypoxia24h to proceed follow-up experiments.Compared with normoxic group,the hypoxia for24h group Mfn2expression lowered, p-Akt expression increased and with the activation of PI3K/Akt signaling pathway, more cells enter the cell cycle of S+G2/M phase.And the PCNA protein expression was increased and mitochondrial apoptosis pathway is inhibited which down-regulate the ratio of cytochrome C in cytoplasm to mitochondrial and the cleaved caspase9protein expression.At last,the PASMCs apoptosis was inhibited;(4)Under hypoxic conditions,LY294002or over-expression group compared with the hypoxia group, Mfn2protein expression increased,p-Akt expression reduced and followed by PI3K/Akt signaling pathway being inhibited, the cell cycle arrested in G0/G1phase,PCNA expression reduced.Then mitochondrial apoptosis pathway was activated,the ratio of cytochrome C in cytoplasm to mitochondrial down-regulated and the cleaved caspase9protein expression increased.At last,the PASMCs apoptosis was activated.(5) While intervene over-expression of the Mfn2and LY294002together does not further enhance the above effect.Conclusion:(1)Under hypoxia conditions, the expression of Mfn2mRNA and in the rats lung tissue of HPH desease model is reduced. The expression of Mfn2protein in PASCMs in hypoxia is also reduced which is following by PASMCs proliferation promoted and apoptosis inhibited.(2)Under hypoxic conditions, down-regulation of Mfn2in PASMCs induced the PI3K/Akt pathway activation and more cells entered into the S+G2/M phase of cell cycle.(3)Under hypoxic conditions, activation of PI3K/Akt signaling pathway by Mfn2down-regulation can further inhibit the mitochondrial apoptosis pathway.Then PASMCs apoptosis was inhibited.