The Role Of SUMO-1in Regulating Of HIF-1a During The Proliferation Of Primary Rat Pulmonary Artery Smooth Muscle Cells In Response To Hypoxia

【Objective】To investigate the effects of hypoxia on the proliferation of primaryculture rat pulmonary arterial smooth cells （PASMCs） and the expression ofSUMO-1、HIF-1α and target genes VEGF； To evaluate the role of SUMO-1inregulating the expression of HIF-1α and VEGF,and the hypoxic proliferation ofPASMCs during the development of hypoxic pulmonary hypertension（HPH）andprovide the theoretical basis of mechanism for HPH.【Methods】 The study consisted of three parts.（1）To separate rat PASMCs andprocess by hypoxia：rat PASMCs were separated by the method of tissue blockanchorage and primary cultured. The morphology of cellular was observed under lightmicroscope，and were identified by projection electron microscopy，and α-smoothmuscle actin（α-SM-actin）immuchemistry. The good growth state of3-5generationPASMCs was placed in the three gas incubator which included1%O2、5%CO2、94%N2,and cultured different times to copy the rat HPH model in vitro.（2）Investigating the effects of hypoxia on rat PASMCs：The primary cultured PASMCswere exposed to normoxic and（or）hypoxic condition for2，6，12，24，48hoursrespectively，then the methods of MTT assay and proliferating cell nuclear antigen（PCNA）immunecellularchemical(ICC) were used to detect the proliferation ofPASMCs. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotwas used to determine the mRNA and protein expression of SUMO-1、HIF-1α andVEGF respectively. Immunofluorescence was used to determine the expressive locationof SUMO-1and HIF-1α under normoxic and hypoxic condition.（3）After geneinterference by SUMO-1， investigate the effects of hypoxia on rat PASMCs：constructing the lentivirus vector transfers PASMCs which containing SUMO-1and SUMO-1siRNA, then divided into four groups randomly,including blank controlgroup、No-load lentivirus group、SUMO-1-RNAi-LV group、SUMO-1-LV group. Theywere exposed to normoxic and hypoxic condition for12hours respectively，RT-PCRand WB was used to determine the mRNA and protein expression of HIF-1α and VEGFrespectively, The above four groups were exposed to hypoxic condition for0、2、6、12、24、48hours respectively，Brdu infiltration law was used to determine the effectsof hypoxia on the proliferation of PASMCs.【Results】1. To separate and identify rat PASMCs：（1）The adherent method of tissue explants separated rat PASMCs successfully. Thecells was primary cultuled by DMEM/F12culture-medium that including1%Penicillin-streptomycin and fetal bovine serum. A few cells freeded from the the aroundof organization block after3-5d, the cells merge into a typical "peak-valley" shapedistribution after8-10d, then digestived and posterity to the fifth generation.（2）Under inverted phase contrast microscope, the cellulars tend to be longspindle,and have oval nuclei at the core of then, forming the “peak-valley” mode.（3）The α-SM-actin of ICC showed, the endochylema was stained in brownish yellow,and the the positive rate was beyond96%；the α-SM-actin of IF showed, there arestrong red fluorescent in the endochylema, that parallels with the vertical axis,but thenuclei aren’t shaded. The above show the cells are PASMCs.（4）Under projection electron microscopy, both the cells and the nuclei were Longspindle；there were a large number muscle cells silk that parallels with the vertical axisand a little dense spot and dense boby under myolemma, but lack of golgiosome.2. the effects of hypoxia on rat PASMCs：（1）The results of MTT assay showed：under normoxia and hypoxia, the absorbance（A values） of PASMCs increased along with the extension of the time, and the Avalues expose to hypoxia were above that of nomoxia；comparing with the distinctionamong the groups of hypoxia, the A values that exposed to hypoxia increased after12h(p＜0.05), reached peek value after24h(p＜0.05), steped in Platform period after48h.（2）The results of ICC showed：the positive of PASMCs increased along with the extension of the time exposed to hypoxia, the positive rate of PASMCs that exposed tohypoxia increased after12h, compared with control group(p＜0.05), reached peekvalue after24h(p＜0.05), steped in Platform period after48h.（3）The mRNA expression of SUMO-1and VEGF in PASMCs exposed to hypoxiaincreased after2h (p＜0.05), reached peek value after12h(p＜0.05), steped in Platformperiod after24h；the mRNA expression of HIF-1α in PASMCs increased along with theextension of the hypoxic time, wheas has No significant differences compared withcontrol group（P＞0.05）.（4）The protein expression of SUMO-1、HIF-1α and VEGF in PASMCs increasedalong with the extension of the time exposed to hypoxia, and reached peek value afterhypoxia12h(p＜0.05), steped in Platform period after hypoxia48h.（5）SUMO-1located in nuclei of PASMCs both under normoxia and hypoxia, wheasHIF-1α of PASMCs expressed trifling in cytoplasm under normoxia, and obviouslyexpressed in nuclei under hypoxia.3. After gene interference by SUMO-1, the effects of hypoxia on rat PASMCs：（1）The mRNAand protein expression of SUMO-1、HIF-1α and VEGF in PASMCs,were significantly lower in SUMO-1-RNAi-LV group than those in controls（P＜0.05）,wheas significantly lower in SUMO-1-RNAi-LV group than those in controls（P＜0.05）,and these were more remarkable when exposed to hypoxia（P＜0.05）.（2）The results of Brdu showed：the positive rate of PASMCs increased along with theextension of the time exposed to hypoxia. The positive rate in PASMCs, weresignificantly lower in SUMO-1-RNAi-LV group than those in controls，especially whenexposed to hypoxia for12h、24h、48h（P＜0.05）；wheas significantly lower inSUMO-1-RNAi-LV group than those in controls especially when exposed to hypoxiafor6h、12h、24h、48h（P＜0.05）.【Conclusion】1. Hypoxia can promote the proliferation of PASMCs obviously.2. Under hypoxia, HIF-1α from the cytoplasm of PASMCs transferred to the nuclei,its transactivational activity and stability was up-regulated by SUMO-1whichexpressed in nuclei raised HIF-1α. 3. Under hypoxia, SUMOylation of HIF-1α up-regulats its transactivational activityand stability by SUMO-1, leading to activated the transactivation of HIF-1α targetgenes such as VEGF, and being thus implicated in the hypoxic proliferation ofPASMCs.