The Protective Role Of VEGF-B Against Cardiac Ischemia-reperfusion Injury And Its Underlying Mechanisms

Background:Ischemic heart disease(IHD)is a major cause for deaths in the contemporary world.At present,some vascular intervention techniques such as percutaneous coronary intervention(PCI)are widely used in clinic so as to restore the cardiac perfusion and thus rapidly alleviate ischemia.Such interventions make patients feel markedly better subjectively;however,the perfusion restoration may be followed by ischemia-reperfusion(I/R)injury and even vascular restenosis.Thus,it is of great significance to prevent I/R injury and vascular restenosis effectively in IHD treatment.Unfortunately,no therapy now available is universally acknowledged to be specially effective.VEGF-B has long been regarded as an ambiguous member of the vascular endothelial growth factor family,and can bind to VEGFR-1 and co-receptors for NRP-1.Up to date,the reports on the effects of VEGF-B on the heart from home and abroad are mainly focused on its transgenic methods.Transgenic research shows that VEGF-B can regulate the vasculature development and the growth,metabolism and survival of cardiocytes in the heart.Yet the practical application of transgenic methods is quite controversial due to people's concern about the safety and controllability of the carriers.Our previous study demonstrated that cardiac stem cells(CSCs),which show traits of VEGFR1 expression,were partly involved in VEGF-A-induced CSC migration,and contributed to the repair of the infarcted heart by inducing angiogenesis.However,it is unclear whether VEGF-B can repair the infarcted heart by VEGFR1-expressing CSCs inducing angiogenesis in the I/R injured heart.And,up to now the effect of VEGF-B on the short-and long-term prognosis following the development of cardiac I/R,myocardial infarction and heart failure is also unclear.Considering that clinical application of human VEGF-B by injection is more acceptable to patients than by transgenic methods,it is of great practical significance to elucidate the role and mechanism of recombinant human VEGF-B protecting against cardiac I/R injury and to clarify the effect of VEGF-B on short-and long-term prognosis of patients suffering myocardial ischemia and heart failure,which may provide experimental groundwork for clinical use of VEGF-B.Objective:To investigate the short-term protective effects of recombinant human VEGF-B on in vivo rat hearts undergoing I/R injury and in vitro H9c2 cells undergoing hypoxia-reoxygenation(H/R)injury,the association of VEGF-B induced angiogenesis with CSCs,and the long-term role of VEGF-B in protecting I/R injured hearts,as well as their potential mechanisms.Methods:(1)In vivo using the model of left anterior descending coronary artery ligation for 1 hour following loose ligation,short-term heart I/R model were established 1 day after reperfusion.Adult male rats were randomly divided into sham operation group,I/R injury group,low dose VEGF-B treatment group(1.0?g/kg)and high dose VEGF-B treatment group(10.0?g/kg).After loose ligation,local injection of recombinant human VEGF-B were exerted.One day after reperfusion,the cardiac hemodynamic parameters were firstly recorded and analyzed using BL420S biological signal acquisition system to study the effect of VEGF-B on the function of the I/R injured heart.And then,the effect of VEGF-B on myocardial infarct and non-infarct areas after I/R injury was observed by 1%TTC immunostaining of the myocardial tissue.Subsequently,the effect of VEGF-B on the activities of chemicals specific to myocardial injury such as CK,cTnT and CK-MB were detected by colorimetry and enzyme-linked immunosorbent assay.Finally,the effects of VEGF-B on cells apoptosis and autophagy in I/R injured myocardial tissues were analyzed by TUNEL and Western blot.(2)In vitro using the model of hypoxia-reoxygenation(H/R)of H9c2 cells,after treated with or without VEGF-B,the LDH levels were firstly determined by colorimetric method to compare the damage of cells from different groups.Then,the effects of VEGF-B on H9c2 cells apoptosis exposed to H/R and on apoptosis-related signaling pathway were analyzed by DAPI staining,Annexin V/PI flow cytometry and Western blot.Next,Ad-mRFP-GFP-LC3 system and Western blot analysis were utilized to observe the traits of autophagic flux with or without VEGF-B in the model of H/R-induced H9c2 cells injury.Subsequently,using autophagic inducer or blocker,the effects of VEGF-B on H/R-induced H9c2 cells apoptosis were explored when inducing or blocking autophagy.Finally,after blocking related signaling pathway,the effects of VEGF-B on H/R-induced H9c2 cells autophagy were explored when blocking PI3K/Akt or p38MAPK signaling pathways by using LY294002 or SB203580,respectively.(3)In vivo using the model of left anterior descending coronary artery ligation for 1 hour following loose ligation,long-term heart I/R model were established after reperfusion for 7 day.The hemodynamic parameters of the rats from each group were firstly recorded using BL420S biological signal recording system to analyze the effect of VEGF-B on cardiac function after in vivo I/R injury.And then,the effects of VEGF-B on CSCs mobilization,angiogenesis and neovascularization were detected by immunofluorescence staining for c-Kit,a-SMA and vWF ?,and Western blot for SDF-la and HGF.At last,In vitro using condition medium from H9c2 cells stimulated by VEGF-B,the effect of VEGF-B on in vitro tube formation of c-kit positive CSCs were observed,and SDF-la receptor and HGF receptor inhibitors were used to confirm the relationship between CSCs-mediated tube formation and SDF-1?/HGF.Results:(1)One day after heart I/R,local injection of recombinant human VEGF-B significantly reduced the area of cardiac infarct in the heart of rats compared with that from I/R group;This treatment also markedly improved the function of the left ventricle,high dose(10?g/kg)VEGF-B treatment had a more pronounced effect than low dose(10?g/kg)VEGF-B treatment;VEGF-B effectively alleviated the I/R injury of the rat heart by inhibiting the activities of CK and CK-MBC and lowering the level of cTnT;Cell apoptosis assay using TUNEL method demonstrated that VEGF-B markedly lowered the apoptosis incidence in the cardiac I/R model.Western blot analysis showed that Bcl-2 expression was obviously enhanced in VEGF-B treatment groups compared with the I/R group;The ratio of LC3-?/? increased in the I/R group compared with that in the sham operation group.Compared with I/R group,both VEGF-B treatment groups showed lowered ratio of LC3-?/?,with the high dose VEGF-B treatment showing a better effect;obvious increase in pAkt level was detected in both the low dose(1?g/kg)and high dose(10?g/kg)VEGF-B treatment groups.Meanwhile the level of pp38MAPK was also lowered in these two groups.High dose VEGF-B treatment was more effective in elevating pAkt level and lowering pERK1/2 and pp38MAPK levels than lose dose VEGF-B treatment.(2)In vitro using H/R induced H9c2 cell damage model,application of VEGF-B remarkably inhibited LDH release and decreased Bax levels while increasing Bcl-2 levels in dose-dependent manner.Simultaneously,VEGF-B obviously suppressed H/R-induced apoptosis and autophagy of H9c2 cells,dose-dependently elevating the pAkt level markedly and pERK1/2 level slightly,and obviously lowered the levels of pp38MAPK.SB203580 and LY294002 markedly canceled the anti-apoptotic effect of VEGF-B,which reveals that VEGF-B prevented cardiocyte apoptosis via PI3K/Akt and p38MAPK signaling pathways;LY294002 blocked the VEGFB-mediated alteration in the expression of Bcl-2 and Bax,indicating that VEGF-B regulated the expression of Bcl-2/Bax via PI3K/Akt signaling pathway.Meanwhile,VEGF-B inhibited the expression of LC3 and Beclinl as well as LC3-?/? rate.The special effects of VEGF-B on autophagy could be abolished by LY294002 and SB203580.Importantly,rapamycin,an autophagy inducer,reversed the VEGF-B-mediated anti-apoptotic effects,the VEGF-B mediated anti-autophagic effects and the LC3-?/? ratio.Conversely,Chloroquine,an autophagy inhibitor,potentiated the VEGF-B-mediated anti-apoptotic and anti-autophagic effects.These results indicated that VEGF-B inhibited H/R induced cell apoptosis via inhibition of autophagy;H/R increased the expression of autophagy related proteins LC3 and Beclinl in H9c2 cells.(3)After 7 days of treatment with VEGF-B,the function of the left ventricle of the heart was significantly improved,with a dose dependent increase in LVSP and+dp/dtmax and decrease in LVEDP.VEGF-B,in a dose dependent manner,increased the expression of SDF-la and HGF in infarcted myocardium,trigering the mobilization of CSCs and the increased number of CSCs in infarcted myocardium,resulting in the increased vessel density through promoting the formation of vWF?positive microvessels and a-SMA positive arteries.In vitro,condition-medium from H9c2 cells stimulated by VEGF-B induced the formation of CSC-mediated tubes,and the specfic effects could be respectively abolished by SDF-la and HGF receptors blockers,indicating that VEGF-B improved heart function through the involvement of activating CSC mediated by SDF-la and HGF in angiogenesis.Conclusion:After cardiac I/R injury for 24 h,recombinant human VEGF-B protein plays a role in autophagy inhibiton-mediated anti-apoptotic effects by regulating PI3K/Akt and p38MAPK signaling pathway in myocardial I/R injury.A week after I/R induced infarction,recombinant human VEGF-B protein triggers CSC mobilization through inducing SDF-1 and HGF expression,and CSC mobilization mediated angiogenesis and vasculogenesis.Therefore,VEGF-B improves the structure and function of the I/R heart through short-term cardiomyocyte protection and long-term activation of CSCs.