EIF2? Promotes Monocrotaline-induced Pulmonary Vascular Remodeling Via Activating Autophagy

? Background ? Pulmonary arterial hypertension(PAH)is a multifactorial and progressive malignant cardiovascular disease.Its main characteristic manifestation is an increase in pulmonary vascular resistance and pulmonary artery pressure,which ultimately leads to right heart failure and even death.Pulmonary vascular remodeling is an important pathophysiological mechanism in PAH.Our previous study found that eukaryotic initiation factor 2?(eIF2?)was involved in pulmonary artery in hypoxia-induced pulmonary hypertension(PH),however,its mechanism is not yet clear.Studies have shown that autophagy was actived in pulmonary vascular remodeling in PAH induced by monocrotaline.Because eIF2? plays an important role in hypoxia-induced PH vascular remodeling,autophagy is also involved in the regulation of PAH pulmonary vascular remodeling.Therefore,we hypothesized that eIF2? may play an important role in monocrotaline-induced PAH pulmonary vascular remodeling by autophagy.?Aims?1.To confirm the important role of eIF2? in the vascular remodeling of PAH induced by monocrotaline;2.To clarify whether eIF2? promote monocrotaline-induced PAH vascular remodeling via autophagy pathway.?Main methods?1.Animal experiment: 150~200g SD rats were induced by intraperitoneal injection of monocrotaline 60 mg/kg,which can be modeled at 4 weeks.After 4 weeks,the mean pulmonary artery pressure(mPAP)and right ventricular systolic pressure(RVSP)were measured by right external jugular vein catheterization.The right ventricle(RV),left ventricular ventricular septum(LV+S)were separated and weighed,and the length of the tibia was measured.Calculate the ratio of RV and(LV+S)and tibia length to right ventricle;some lung tissues were fixed with 5% formaldehyde to observe pulmonary vascular remodeling by paraffin embedding;the proliferation markers Ki67 and PCNA mRNA expression was detected by Real-time PCR;the mRNA expression of eIF2? was detected by Real-time PCR;the protein expression of p-eIF2?,eIF2?,LC3 B,p62 were detected by Western-blot.2.Cell experiment: The primary PASMCs were taken from the SD rats,and the third generation cells were used for immunohistochemistry(?-actin)to identify cells,and the third to eight generations cells were used for experiments.PASMCs proliferate were induced by PDGF,and the proliferation was detected by CCK-8.confirming the important regulation of eIF2? on autophagy and PASMCs proliferation by eIF2? siRNA technology.Chloroquine treatment of PASMCs inhibited autophagy to confirm that autophagy mediates eIF2? induced PASMCs proliferating.The mRNA expression of Ki67 and PCNA was detected by Real-time PCR,and the expression of eIF2?,LC3 B and p62 protein was detected by Western-blot.?Results?Compared with in the normal control group,(1)In the animal model of PAH induced by monocrotaline,the levels of mPAP and RVSP were significantly increased in rats,and the ratio of RV/LV+S and the length of right ventricle to tibia was significantly increased.HE staining showed significant remodeling of pulmonary angiogenesis and vascular intima thickness.The percentage also increased significantly,indicating that monocrotaline induced significant pulmonary vascular remodeling in PAH;(2).The results of Real-time PCR showed that the expression of proliferation markers Ki67 and PCNA mRNA was increased,indicating that the proliferation of PASMCs induced by monocrotaline in PAH was obvious.The above indicates that the monocrotaline-induced PAH model was successfully established.(3).The expression of eIF2? mRNA in rat pulmonary arteries was increased in PAH model.Western-blot results showed that the expression of p-eIF2?,eIF2? and LC3 B protein was significantly increased in monocrotaline-induced PAH,while the expression of p62 protein was significantly decreased in monocrotaline-induced PAH,indicating that autophagy was activated.(4).In PDGF induced the proliferation of PASMCs,the expression of eIF2? and LC3 B protein was significantly up-regulated and the expression of p62 protein was down-regulated,indicating that autophagy was activated;eIF2? siRNA inhibited the up-regulation of eIF2?,LC3 B protein expression and down-regulation of p62 protein expression induced by PDGF;(5).eIF2? siRNA inhibits proliferation of PASMCs induced by PDGF.(6).Chloroquine caused a significant increase in LC3 B,p62 protein expression and inhibited the proliferation of PASMCs caused by PDGF.?Conclusions?eIF2? can promote PASMCs proliferation and PAH pulmonary vascular remodeling via autophagy activation,and targeted inhibition of eIF2? can provide new targets for the treatment of PAH diseases.