The Role Of SENP-1Regulate Hypoxia-inducible Factor-1α In Hypoxia-induced Proliferation Of Rat Pulmonary Artery Smooth Muscle Cells

【Objective】Hypoxic pulmonary vascular remodeling (HPSR) plays an important rolein the development of hypoxic pulmonary hypertension (HPH).The main mechanism ofthe HPSR is mediated by the proliferation of pulmonary artery smooth muscle cells(PASMCs) and the molecular basis is the signal pathways which is mediated byHIF-1alpha.This study was to research on the effects of hypoxia on the proliferation ofthe PASMCs and the expression of SENP-1, HIF-1alpha and downstream responsivegenes VEGF in the PASMCs when they were exposed to different hypoxia times.Toinvestigate SENP-1whether regulate the HIF-1α-VEGF signaling pathway underhypoxia and hypoxia-induced proliferation of PASMCs,then provide a new theoreticalbasis for the pathogenesis of HPH.【Methods】 The experiment consists of four parts.（1）To extract rat PASMCs and expose to hypoxia：The PASMCs were successfullyextracted by making the tissue block of the tunicae media vasorum adhered to the wallof the flasks.The rat PASMCs were primary cultured and subcultured well.Under theinverted phase contrast microscopic,we observed the morphology of the cells.Toidentified whether they were smooth muscle cells by using immunofluorescence ofα-smooth muscle actin（α-SM-actin）antibodies.The3rd or4th generation grew wellcells were putted into the three-gas incubator (containing1%O2,5%CO2,94%N2),and exposed to hypoxia for different times, grouped them by hypoxia duration.（2）To research on the effects of hypoxia on the proliferation of the PASMCs：Thecells in logarithmic growth phase,after cell cycle synchronization,were exposed tonormoxic or hypoxic conditions for2h，6h，12h，24h，48h respectively.The proliferationof rat PASMCs was detected by Cell Counting Kit-8and EdU assay.（3）To research on the effects of hypoxia on SENP-1、HIF-1α and VEGF in rat PASMCs：The PASMCs, in logarithmic growth phase,were randomly divided intonormoxic group and hypoxic (2,6,12, and24) h group.The mRNA and protein level ofSENP-1、HIF-1α and VEGF was detected by RT-PCR and western blot respectively.（4）Knocked down the SENP-1gene,then detected the effects of hypoxia onPASMCs：Constructed the lentivirus vector infected PASMCs which contained siRNAthat can knocked down the SENP-1gene specifically, then divided the PASMCs intothree groups randomly,which including blank control group、blank lentivirus group andsiRNA intervention group.These groups were exposed to normoxic and hypoxicconditions for12hours respectively.The mRNA and protein level of HIF-1α and VEGFwas detected by RT-PCR and western blot. EdU Immunofluorescence was used todetect the effect of hypoxia on the proliferation of PASMCs when SENP-1wasknocked down.【Results】1. To extract and identify rat PASMCs：（1）Selected weighing about150-180g male SD rats,then separated pulmonaryvascular and removed the inner and outer membranes.Tissue adherence method wasused to separate the PASMCs.After3-5days,there were some cells freeded from thetissue block.They are long spindle cells and when they fused to80%-90%,then purifiedthem as used the method of differential adhesion speed.Subcultured the PASMCs,thepurity of the cells can reach to90%.（2）Under the inverted phase contrast microscope,we could see lots of long spindlecells with oval nuclei at the core of cytoplasm.The cells grew well can merge into thetypical "peak-valley" shape distribution.Immunofluorescence staining of smoothmuscle-specific antibody the α-SM-actin showed, there were strong green fluorescentin the cytoplasm,but the nuclei are not colored. The above experimental results showedthat the cells which we separated were PASMCs.2. The effects of hypoxia on the proliferation of the PASMCs：（1）The results of CCK-8showed：under normoxic and hypoxic conditions, the opticaldensity（OD values）of the PASMCs increased along with the extension of the time, and the OD values in the hypoxic condition were more than in the normoxiccondition.Compared the distinction among the hypoxic groups,we could see the ODvalues of the PASMCs that exposed to hypoxia increased after12h (p＜0.05),after24hit reached high value (p＜0.05).The OD value of the hypoxia48h group was no longerto increase.（2）The results of EdU Immunofluorescence showed：with the hypoxia timeprolonged,the EdU marked cells increased.Compared the number of positive cells andthe ratio of proliferating cells, the two indicators of the hypoxia12h group weresignificantly higher than control group (p＜0.05).The positive cells and positive ratiosreached high value after24h(p＜0.05), then steped in platform after48h.3. The effects of hypoxia on SENP-1、HIF-1α and VEGF in rat PASMCs：（1）The results of RT-PCR showed：Compared with normoxic group,the mRNAlevel of SENP-1and VEGF in rat PASMCs which exposed to hypoxic conditions for2hours was already increased obviously (p＜0.05).With hypoxia time prolonged themRNA expression of the two genes was increased,and hypoxia12h group was up to themaximum value (p＜0.05).The increasing trend of hypoxia24h group was not obviousthan hypoxia12h group.Hypoxia could stimulate the expression of HIF-1alphamRNA, but the difference between the hypoxia groups was not obvious (P＞0.05).（2）The results of Western blot showed：With hypoxia time prolonged,the SENP-1,HIF-1alpha and VEGF protein expression levels of the rat PASMCs showed anincreasing trend,and reached maximum value after hypoxia12h(p＜0.05), then reacheda plateau of development after hypoxia48h.4. After knocking down the SENP-1gene, the effects of hypoxia on rat PASMCs：（1）The results of EdU showed：among blank control group、blank lentivirus groupand siRNA intervention group,compared the number of positive cells and the ratio ofproliferating cells the hypoxia12h groups were higher than the control groups.Thepositive ratio in PASMCs, were significantly lower in siRNA intervention group thanthose in control groups(p＜0.05). （2）The mRNAand protein level of SENP-1、HIF-1α and VEGF in PASMCs, werelower in siRNA intervention group than those in control groups(p＜0.05),and theresults were more remarkable when exposed to hypoxia(p＜0.05).【Conclusion】（1）Hypoxia could significantly promote the proliferation of the rat PASMCs,whichcultured in vitro,and the signaling pathway which HIF-1alpha regulated was theimportant molecular basis in this process.（2）In hypoxic condition,HIF-1alpha could be deSUMOylation mediated by SENP-1which could up regulate its transactivational activity and stability.It could lead toactivate VEGF,which was one target gene of the signaling pathway of HIF-1alpha,thenplay an important role in the hypoxic-induced proliferation in rat PASMCs.