PDGF Prmotes Warburg Effect In Pulmonary Arterial Smooth Muscle Cells Via Activiation Of PI3K/AKT/mTOR/HIF-1α Signaling Pathway

Background and purpose pulmonary arterial hypertension(PAH)is characterized by progressive pulmonary circulation resistance increases and pulmonary arterial remodeling,and eventually leads to right heart failure and death.Pulmonary artery smooth muscle cells(PASMCs)abnormal proliferation and migration is central part of pulmonary arterial remodeling,inhibit abnormal proliferation and migration of PASMCs effectively reverse pulmonary arterial remodeling and decrease pulmonary resistance.Recent studies have shown that the Warburg effect play an important role in pathological of the PAH,moreover,it remains unclear whether the Warburg effect is involved in PASMC proliferation.Platelet-derived growth factor(PDGF)is a receptor tyrosine kinase,and studies have found that PDGF and platelet-derived growth factor receptor(PDGFR)signaling pathways involved in the Warburg effect in tumor cells,at the same time,studies also have shown that increased expression of PDGF and PDGFR were found in lung tissue of patients and animals with PAH.,and PDGF promoting PASMCs proliferation along with increased lactate production.Thus,we hypothesized that the Warburg effect mediates PASMC proliferation induced by PDGF.We separated culture PASMCs from normal SD rats PASMCs,and inhibited PDGFR,PDGFR signaling pathway,and Warburg effect in PDGF-induced PASMC proliferation.Understanding the Warburg effect whether mediates PDGF promoting PASMCs proliferation,and understanding its signaling pathways by which regulate the Warburg effect in PASMCs,and revealing the mechanism and roles of the Warburg effect in the pathological of PAH.Methods(1)The PASMCs cultured in vitro with type I collagenase.Before the PASMCs cultured,SD rats pulmonary trunk separated were subjected to outer membrane peel and endothelial cells remove by enzymatic digestion in a sterile environment.We observed the status and characteristics of PASMCs with inverte phase contrast microscope,determined the cell viability with trypan blue staining,and identified the α-smooth muscle actin(α-SM actin)with immunocytochemistry staining.(2)Treatment PASMCs with different concentrations(0,5,10,15 ng/mL)of PDGF for different processing time(0,6,12,24 h),cell viability,proliferating cell nucleus antigen(PCNA)protein expression were determined by MTT assay and western blot.(3)PDGFR antagonists(sorafenib,imatinib)were used in pretreatment of the 15 ng/m L PDGF-treated PASMCs from SD rats.In addition,the Warburg effect inhibitors 2-DG and DCA were used as positive controls.Levels of extracellular glucose consumption,lactic acid(LD)and cellular ATP were determined by glucose assay kit,and LD assay kit,or an ATP assay kit.Metabolic enzyme activities assay,such as Glut1,Glut4,MCT4,PDH,LDH,and PCNA,were determined by western blot.Cell viability was performed by MTT assay.(4)PDGFR antagonists(sorafenib,imatinib),the Warburg effect inhibitors(2-DG,DCA),PI3 K inhibitor(LY294002)and mTOR inhibitor(RAPI)were used in pretreatment of the 15 ng/mL PDGF-treated PASMCs from SD rats.Levels of extracellular glucose consumption,lactic acid(LD)and cellular ATP were determined by glucose assay kit,and LD assay kit,or an ATP assay kit.Expression of Glut1,Glut4,MCT4,PDH,LDH,PCNA,PDK1 HIF-1α,and PI3 K,AKT,mTOR protein expression and its phosphorylation level were determined by western blot.Cell viability was performed by MTT assay.Results(1)The cultured PASMCs subjected to enzymatic digestion and isolation presented shuttle shape at day 3,typical peak-valley-like growth at day 6,and 90% confluence at day 9.The morphological observation and immunocytochemistry staining identification showed that: the cells cultured were PASMCs.(2)PDGF increased PASMCs viability and expression of PCNA protein with increasing of PDGF concentration(P < 0.05).PDGF increased PASMCs viability and expression of PCNA protein with increasing PDGF treatment time(P < 0.05).(3)Compared with the group without PDGF,15 ng/mL PDGF markedly increased the extracellular glucose consumption,lactate production,and expression of Glut1,Glut4 MCT4,LDH(P < 0.05),moreover,significantly decreased intracellular ATP production and expression of PDH(P < 0.05);Compared with PDGF treatment groups,PDGF inhibitor significantly suppressed extracellular glucose consumption,lactate production,and expression of Glut1,Glut4 MCT4,LDH(P < 0.05),and significantly increased intracellular ATP production and expression of PDH(P < 0.05);Compared with PDGF treatment groups,PDGF inhibitors and Warburg effect inhibitors significantly suppressed PASMCs proliferation induced by PDGF(P < 0.05).(4)Compared with the group without PDGF,15 ng/mL PDGF markedly increased PI3 K,AKT,mTOR phosphorylation levels(P < 0.05);Compared with PDGF treatment groups,PDGFR inhibitors significantly inhibited PI3 K,AKT,mTOR phosphorylation levels induced by PDGF(P < 0.05),however,Warburg effect inhibitors have no obvious inhibited effect in PI3 K,AKT,mTOR phosphorylation levels induced by PDGF;Compared with PDGF treatment groups,PI3 K and mTOR inhibitors remarkably attenuated the extracellular glucose consumption,lactate production,expression of Glut1,Glut4 MCT4,LDH,and expression of HIF-α,PDK1(P < 0.05),moreover,significantly increased intracellular ATP production and expression of PDH(P < 0.05);Compared with PDGF treatment groups,PI3 K and mTOR inhibitors markedly suppressed PASMCs proliferation induced by PDGF(P < 0.05).Conclusions1.PDGF promotes PASMC proliferation accompanied by enhanced Warburg effect.2.PI3K/Akt/mTOR/HIF-1α signaling pathway mediates the Warburg effect in the proliferative PASMCs.