RELM-βRegulates Ca²⁺/PI3K/Akt/mTOR Signaling Pathway In Hypoxia-induced Proliferation Of Human Pulmonary Artery Smooth Muscle Cells

【Objective】To observe the effect of hypoxia on human pulmonary artery smoothmuscle cells (PASMCs) cultured in vitro,and RELM-β expression in human PASMCswhen they were exposed to different hypoxia times;To explore the role of RELM-β inhuman PASMCs proliferation and the expression of [Ca2+]ipathway-PI3K/Akt/mTORwhen added recombinant RELM-β proteins, then provide a new theoretical basis forpulmonary vascular remodeling (HPSR).【Methods】Experiment was divided into four parts.（1）Extract human PASMCs and expose them to hypoxic: the human PASMC wereextracted by using some isolated tissue block of the tunicae media vasorum adhered tothe wall of the flasks. Cells morphology were observed under phase contrastmicroscopic, and identified by using cellular immunology of α-smooth muscle actin(α-SM-actin). The cells were placed in37℃,1%O2,5%CO2,94%N2three gasincubator,and exposed to differen hypoxia time(0h,3h,6h,12h,24h and48h).（2）To detect the effects of hypoxia on the proliferation of human PASMCs andRELM-β expression in hypoxic conditions:the cells of3-5generations were cultured byDMEM/F12medium without FBS cells24h after the cycle synchronization,then exposdto the normoxic or hypoxic conditions for3h,6h,12h,24h,48h.The proliferation wasdetected by Cell Counting Kit8(CCK-8).The RELM-β mRNA and protein weredetected by real-time quantitative detection of nucleic acid amplification (RealTime-PCR) and Western blot (Western blot).（3）To research the relation of RELM-β and human-PASMCs proliferation: using EdU to detect the human PASMCs proliferation in different concentrations ofrecombinant protein RELM-β (group A (0ng/ml), group B (5ng/ml), group C(10ng/ml),group D (20ng/ml), group E(30ng/m).（4）To research the expression of Ca2+signaling pathway-PI3K/Akt/mTOR inhuman PASMCs when adding recombinant human protein RELM-β:Cells arerandomly divided into four groups:1, control group;2, after added the reorganizationRELM-β20ng/ml30min;3, after treated with EGTA (extracellular calcium chelator)30min, then added the reorganization RELM-β20ng/ml in30min;4,after treated withBAPTA (intracellular calcium chelator)30min, then added the reorganization RELM-β20ng/ml in30min.On the other hand, after added PI3K’s inhibitor LY294002,cellswere divided into four groups:1, control group;2, only added recombinant humanprotein RELM-β10ng/ml;3,after treated with LY29400230min, then added thereorganization RELM-β10ng/ml in30min.The PI3K/Akt protein were deceted byWestern blot.【Results】1.Extraction and identification of human PASMCs：（1）selected the pulmonary artery of patients in clinical lobectomy,pulmonary arterywas isolated under sterile conditions and cut into pieces in the film,the isolated humanPASMCs by using tissue adherent successfully.The cells were cultured by DMEM/F12medium containing with1%penicillin-streptomycin and15%FBS.Selected thepulmonary artery of patients in clinical lobectomy,pulmonary artery was isolated understerile conditions and cut into pieces in the film,the isolated human PASMCs by usingtissue adherent successfully.The cells were cultured by DMEM/F12medium containingwith1%penicillin-streptomycin and15%FBS.A little cells freed from the tissueblock after10-12days.The cells were purified by using differential adherent,and thepurity of the cells can be reach to90%.（2）Under the microscope,we could see the cells grow like fusiform radial, abundantcytoplasm and with oval nuclei located in the center of the cell;15-17days after cell fusion into pieces, covered bottom, bundles arranged parallel to the growth of smoothmuscle cells showed typical "peak-valley" shape.Immunofluorescence staining ofsmooth muscle-specific antibody showed that cytoplasmic obviously colored in redfluorescence, distributing along with filaments, and nuclear colored in bluefluorescence by DAPI. So, it can be proved the cells were human PASMCs.2.The effects of hypoxia on the proliferation of human PASMCs and RELM-β（1）The CCK-8results showed:whether under normoxic or hypoxic conditions, theoptical density (OD values) of the cells increased along with the extension of thetime,and the OD values in the hypoxic were more than in the normoxicconditions.Hypoxia12h,24h,48h group with each corresponding normoxic group werestatistically significant (P <0.05); compared with each other in hypoxia,hypoxia12h,24h,48h and hypoxia3h,6h differences were statistically significant (P <0.05),after24h it reached high value (P <0.05).（2）The result of WB and q-PCR showed:Compared with normoxia group,theRELM-β expression in human PASMC which exposed to hypoxic condition for3hours was already increased obviously(P <0.05), after12h it reached high value (P<0.05);it turned to plateau after hypoxia24hours;48h hypoxia reduced but still wasstatistically significant (P <0.05) than normoxic and hypoxic3h.3.The effects of RELM-βon the proliferation of human PASMCsThe results of EdU immunofluorescence showed: with the increasing ofrecombinant protein RELM-βconcentration,the EdU marked cells increased.Comparedthe number of positive cells and the ratio of proliferating cells, the group D (20ng/ml)were significantly higher than control group(P <0.05).4.The PI3K/Akt/mTOR signaling pathway activated by increased Ca2+（1）The expression of PI3K and Akt were significantly higher than control groupafter adding recombinant protein RELM-β (P <0.05); after adding calcium chelator,the expression of PI3K and Akt were reduced, and the difference was statisticallysignificant (P <0.05).（2）After adding PI3K inhibitor-LY294002, the expression of Akt wassignificantly decreased compared with the control group (P <0.05). 【Conclusion】1. Hypoxia can upregulate the expression of RELM-β in human PASMCs, therebypromoting the proliferation of human PASMCs.2. The RELM-βcan reglate the Ca2+/PI3K/Akt/mTOR signaling pathways, it play animportant role in the hypoxic-induced proliferation in human PASMCs.