HIMF Regulate Hypoxia-induced Rat Pulmonary Artery Smooth Muscle Cell Proliferation

【Objective】To study the mechanism for hypoxia-induced mitogenic factor(HIMF) regulationof rat pulmonary artery smooth muscle cells (PASMCs) proliferation, and explore thefunction of Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signaling pathways in thisprocess.【Methods】⑴The rat PSAMCs were primary cultured and randomly divided intonormoxic(control) and hypoxic conditions for3h,6h,12h,24h,48h, and then thelevels of mRNA and protein of HIMF expression every group were tested Real-timeQuantitative PCR and Western blotting respectively;⑵Construction of lentivirus vector of overexpression HIMF, and it was used toobserve its influence on each PASMCs group proliferation by EDU assay;⑶On the one hand, PSAMCs were randomly divided into normal Control group(Group A), Empty plasmid transfection group (Group B), and Overexpression HIMFgroup (Group C).Each group in turn added separately EGTA (extracellular Ca2+chel-ator), BAPTA-AM (intracellular Ca2+chelator), A23187(Ca2+carrier agonist),LY294002(PI3K inhibitor), Akt inhibitor detect to detect the levels of p-PI3k, p-AktThr308, p-Akt Ser473, p-mTOR proteins of every group;⑷On the other hand, the same as above grouping,each group in turnadded,separately,EGTA(extracellular Ca2+chelator),BAPTA-AM(intracellular Ca2+che-lator),A23187(Ca2+carrier agonist),staurosporine (PKC inhibitor), to detect the levels of p-PKC, p-p44/42(Thr202/Tyr204),p-p38(Thr180/Tyr182) proteins of every group.【Results】⑴mRNAand protein levels of HIMF were few expressed in normoxic group,under hypoxic condition, their expression were significantly increased andsignificantly different compared with control group (P<0.05), while they reached thehighest level at both H6h groups;⑵Edu cell proliferation assay results show:Overexpression HIMF group cansignificantly increased the proliferation of PASMCs; and compared with Controlgroup and Empty plasmid transfection group, it was significantly differentrespectively(P <0.05);⑶Compared with Control group and Empty plasmid transfection group, theOverexpression of HIMF group, can increase protein expression levels p-PI3k,p-AktThr308, p-AktSer473, p-mTOR, p-PKC, p-p44/42, p-p38, and the differenceswere statistically significant (P <0.05).【Conclusions】⑴Hypoxia can promote expression level of HIMF protein at rat PASMCs,which can be secreted into the extracellular medium supernatant;⑵HIMF plays a significant role in regulating the proliferation of rat PASMCs;⑶Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signaling pathways were played ansignificantly important role in this process.