The Role And Mechanism Of SIRT6 In Pathogenesis Of Vascular Remodeling Involved In Hypoxia Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension(PAH)is a cardiovascular disease caused by various pathogenic factors,characterized by pulmonary arterial pressure rise and pulmonary vascular resistances increase.The pathological manifestations of PAH are pulmonary vasoconstriction,pulmonary vascular remodeling and the situ thrombosis,then dramatic decline of pulmonary blood flow and severe damage to the right ventricular function,fail the organ and even lead to death in the end.This has great potential danger and affects the patient's quality of life and prognosis seriously.There are many pathogenic factors that lead to PAH,including genetic,congenital,nerve body fluids,hypoxia and so on.Presently,the researches on pathogenesis mechanism of PAH are performed from perspectives of genetic,ion channels,vascular active substances imbalance,inflammation response and the alteration of cellular function.Currently,the therapeutic drugs to PAH comprise phosphodiesterase type 5 inhibitors,endothelial receptor antagonists and prostacyclin analogues.However,because of the limitation to the drug efficacy,only about half of the patients' clinical symptoms and cardiac function improved,and most of the patients will gradually develop drug resistance to the current targeted therapy drugs,leading to decline and even vanishing therapeutic effect.Hypoxia plays an important role in the development of PAH.Under hypoxia condition,pulmonary arteriole contracted,causing the elevation of pulmonary vascular pressure,eventually resulting in the right ventricular pressure.However,when exposed to hypoxia for a long time,serious pathogenesis changes would happen in the pulmonary arteriole,including the apoptosis decrease and enhance the proliferation of PASMCs(pulmonary arterial smooth muscle cells),intimal thickening and form cluster damage,these would cause higher pulmonary arterial pressure,and the failure of right ventricular function and pulmonary vascular remodeling would follow.Under the condition of hypoxia,pulmonary vascular remodeling is the key symbol of PAH,and the histopathology is mainly characterized by the media membrane smooth muscle hypertrophy.However,pulmonary arterial media membrane thickening is resulted from hypoxia induced abnormal proliferation of PASMCs.Therefore,further study on the cell and molecular mechanisms of pulmonary vascular remodeling will become the main purpose of this research.In the normal differentiated cells,the energy mainly comes from glucose aerobic metabolism,but in the tumor cells,the energy mainly relies on glycolysis.Cancer cells always change energy metabolism to glycolysis,even when oxygen is abundant.This phenomenon was called Warburg effect and firstly demonstrated by Otto Warburg,a German biochemist,in the 1920s.The energy metabolism changed from aerobic phosphorylation to aerobic glycolysis was mainly mediated by HIF transcript factors.Therefore,in the tumor cells,by activating the expression of HIF could mediate a series of genes related to glycolysis,angiogenesis and cell survival,including glucose transport(GLUT),vascular endothelial growth factor(VEGF),erythropoietin(EPO)and some glycolytic enzymes,then promoting the happening of the Warburg effect.At present,many studies found that the pathogenesis of PAH is similar to cancer,they all showed the Warburg effect characterized by glucose metabolism type conversion.Therefore,it would be a new therapy direction for the treatment of PAH from the perspective of energy metabolism to study the mechanism of pulmonary vascular remodeling.Hypoxia induced factor(HIF)is the key transcript factor in Warburg effect.HIF has three subunits,includes HIF-1,HIF-2 and HIF-3.HIF-1is a heterodimer that consists of HIF-1? and HIF-1?,HIF-1? is the base of oxygen reaction and HIF-1? is the expression base.Under normoxia conditions,the two proline residues of HIF-1?was hydroxylated through the catalysis of HIF-1 prolyl hydroxylase(PHD1,2,3),then identified by the ubiquitin proteasome ligase von hippel landau(VHL),made HIF-1? ubiquitin and subjected to proteasomal degradation.During hypoxia,the hydroxylation of HIF-1? was suppressed,and accumulation in the nucleus,then HIF-1? dimerized with HIF-1? and bind to the hypoxia responds elements(HREs),resulting in the transactivation of downstream target genes.All of these are related with metabolism contain GLUT1,lactate dehydrogenase(LDH),pyruvate dehydrogenase(PDK).Under hypoxia conditions,the improved expression of HIF-1? could up-regulate the expression,and then the activation of PDH was suppressed,leading to metabolism converse to glycolysis,and the happening of Warburg effect.Silent information regulator 6(SIRT6)is a member of NAD+-dependent deacetylase sirtuin family,which has the NAD+-dependent histone deacetylases and mono-ADP-ribosyl transferases activation,and mainly resides in the nucleus.It was,not only cause the histone and non histone acylation,but also cause parts of the protein substrate ribosylation.SIRT6 play a critical role in regulating genomic stability,metabolism,inflammation,cells senescence and lifespan extension.Recently,SIRT6 plays role in metabolism pathway that has drew more and more attention from researcher.Research found that SIRT6 functions as a histone deacetylase and to mediate the expression of multiple glycolytic genes.SIRT6-deficient exhibited increased HIF-la activity and glucose uptake with up-regulation of glycolysis and decrease mitochondrial respiration,and maintains blood glucose balance.In addition,SIRT6could inhibit the formation of fatty liver through regulating glucose and fatty metabolism,and knock out the SIRT6 could cause obesity in the mice nervous system.From these results,we could conclude that SIRT6 play an important role in the process of energy metabolism.In this study,we mainly discussed whether SIRT6 through interacting with HIF-1?,inhibition of HIF-la transcriptional activity,thereby impeded the key molecules activation of glucose metabolism pathway,eventually suppressed the Warburg effect and reversed pulmonary vascular remodeling,then improved the prognosis of PAH and provided new therapeutic strategies for the treatment of PAH.Methods1.The culture and identify of human pulmonary arterial smooth muscle cellshuman pulmonary arterial smooth muscle cells(HPASMCs)were incubated in Dulbecco's modified eagle's medium(DMEM)high glucose supplemented with 10%fetal bovine serum(FBS)in a humidified atmosphere of 21%O2,5%CO2 at 37?.HPASMCs were used in all experiments between 4 and 8 passages.When cells achieving 95%confluence,subculture the cells.Immunofluorescence staining was performed to detect the mark protein ?-smooth muscle actin(a-SMA),contributed to identify HPASMCs morphology.2.Construction of hypoxia pulmonary artery hypertension model and correlation indicators detectionHPASMCs were seeded in the culture vessels and incubated in a humidified atmosphere of 1%O2,5%CO2 at 37?.Experiments were divided into normoxia group,hypoxia(6h,12h,24h,36h,48h,72h)group.After all the interventions finished,the cells function experiment were performed:(1)We applied CCK-8 assay to detect the proliferation of HPASMCs and verify HPASMCs proliferation of time-dependent.HPASMCs were seeded in 96-well plates,3000cells/well.They were incubated in normoxia and hypoxia environment respectively.After the intervention finished,removed the medium and rinse the cells with PBS twice,then add 100?l medium with 10?l CCK-8 solution and incubated 2h in incubator at 37?.OD value was test by microtiter plat with 450nm wavelength.(2)The apoptosis rate was test by Flow cytometry.HPASMCs were seeded in 60mm culture dish,2×105 cells/well.They were incubated in normoxia and hypoxia environment respectively.After the intervention finished,removed the medium and rinse the cells with PBS twice.Add 1ml trypsin to the culture dish to digest the cells.When the cells began to shrink gradually,removed the trypsin and the cells were terminate digestion by the culture medium with 10%FBS.Cells were collected and rinsed twice with PBS.Add 5?l Annexin V-FITC into cells and incubated for 15min in dark at 37?,then 5?l 7-AAD reagent were added into the cells.Finally,the apoptosis were detected by Flow cytometric.(3)The protein expression of SIRT6,HIF-la and PDK4 were detected by Western blot.After the HPASMCs were intervened finished,the cells were lysed by RIPA lysis buffer,and then collected the total protein.The protein concentration was measured by BCA kit.The protein levels of SIRT6,HIF-la,PDK4 and a-tubulin were test by western blot.(4)The mRNA expression of SIRT6,HIF-1? and PDK4 were tested by real-time PCR.After the HPASMCs were intervened finished,the cells were lysed by Trizol regent,then the total RNA were collected and the reverse transcription reaction were conducted.The Ct Value of SIRT6,HIF-1?,PDK4 and ?-actin was measured by the PCR instrument.3.The position of SIRT6,HIF-1? and PDK4 were detected by immune-fluorescence staining in HPASMCs.4.Recombinant adenovirus Ad-SIRT6 transfected into HPASMCs and related incators detectionHPASMCs were seeded in 6 well plates,1 ×105 cells/well.When cells achieving 60%confluence,recombinant adenovirus Ad-SIRT6 transfection was conducted,according to the MOI=50,60,75,90,100,added the volume of adenovirus SIRT6.Then the cells were collected,the protein and mRNA levels of SIRT6 were tested by western blot and RT-PCR.5.After transfect the recombinant adenovirus SIRT6,the detection of HPASMCs proliferation was conducted by CCK-8kit and EdU staining.The apoptosis rate and cell cycle of HPASMCs were detected by Flow cytometry and Tunel staining.6.Western blot and RT-PCR were used to test the protein and mRNA expression of SIRT6,HIF-la,PDK4,a-tubulin and ?-actin as internal gene respectively.7.Statistical analysisAll the data are analyzed using SPSS 19.0 statistical software.The results are shown the mean ± standard error(X±SEM).Comparison between groups using One-Way ANOVA;when the variance is together,the least significant difference(LSD)is used;otherwise Dennett's T3 method is used.P<0.05 is considered statistically significant.Results1.HPASMCs were identified by using the special maker ?-SMA of smooth muscle cells and DAPI staining the nucleus,fluorescence microscopy observed the cells structure.The result show that all of the HPASMCs are present ?-SMA positive staining,cell cytoplasm was staining green,and the filaments were found in the cytoplasm.At the time,we found that SIRT6 and PDK4 mainly in the nucleus;HIF-1? in the nucleus and cytoplasm.2.Compared with the normoxia group,hypoxia promoted the proliferation of HPASMCs.At the time of 24h,36h,48,72h,hypoxia significantly increase the proliferation rate of HPASMCs(P<0.05),the proliferation was time-dependent.The apoptosis result show that hypoxia significantly decrease the apoptosis rate of HPASMCs(P<0.05),but there were no significant different between hypoxia groups.With the increase time of hypoxia,the late apoptosis rate is also increased.3.Under hypoxia condition,western blot and RT-PCR were performed to detect the expression changes of SIRT6,HIF-la and PDK4.The results showed as follows:compared to normoxia group,the protein and mRNA expression levels of SIRT6 were obvious down-regulation(P<0.05);however,the expression levels of HIF-1?and PDK4 were up-regulate,at hypoxia 48h,the protein expression of HIF-1? and PDK4 significantly increase(P<0.05).4.After transfect the adenovirus and vector,the apoptosis rate and cells cycle of HPASMCs were tested by Flow cytometry,and the proliferation activation was measure by CCK-8 and EdU staining.We found that:compared with hypoxia and vector groups,overexpression of SIRT6 could significantly reduce the proliferation rate of HPASMCs(P<0.05).Meanwhile,the apoptosis was increase.For the result of cells cycle,we observed that over-expression of SIRT6 arrested cells in G1,the proportion of S state was obviously decrease(P<0.05).5.After transfect the adenovirus and vector into HPASMCs,Western blot and RT-PCR were used to detect the expression of SIRT6,HIF-1? and PDK4.The results showed that over-expression of SIRT6 could significantly decrease the protein and mRNA expression of HIF-1? and PDK4(P<0.05).However,there was no significant difference between hypoxia group and vector group(P>0.05).Conclusions1.Hypoxia promoted the proliferation of HPASMCs,and at hypoxia 48h,the proliferation change was the most significantly,so we confirmed the HPAH model was constructed successfully.2.We demonstrated that SIRT6 was participated in the hypoxia induced pulmonary vascular remodeling.Hypoxia induced the abnormal proliferation of HPASMCs was related to the down-regulation of SIRT6,and the up-regulation of HIF-1? and PDK4.3.Recombinant adenovirus was transfected into HPASMCs successfully,and significantly promoted the up-regulation of SIRT6,resulting in reducing the expression of downstream signaling pathway molecules HIF-1? and PDK4,inhibited the abnormal proliferation of HPASMCs and enhanced the apoptosis,contributed to reverse pulmonary vascular remodeling.