The Effects Of Endothelium-specific CYP2J2 Overexpression On The Attenuation Of Retinal Endothelial Cell Senescence In A Rat Model Of Retinal Ischemia/Reperfusion Injury

BackgroundRetinal ischemia-reperfusion(I/R)injury,which can be induced in the animal model of retinal ischemia and reperfusion,results in retinal ganglion cells(RGCs)loss and vascular damages.A significant RGC loss occurs 2 days after retinal I/R injury.In the meanwhile,there are different types of vascular damages: increased retinal vascular permeability and reduced endothelium-dependent dilation of retinal arterioles also in 2 days after injury,while capillary degeneration happens 7 days after I/R injury.Cellular senescence is a permanent state of cell cycle arrest with cellular viability preserved.It is triggered by excessive extracellular stress(such as oxidative stress)or intracellular stress,which are referred to as premature senescence and proliferative senescence,respectively.Vascular endothelial cell senescence results in abnormalitites in vascular structure and function,such as increased retinal vascular permeability and altered tissue perfusion.Cytochrome P450 2J2(CYP2J2)is one of the CYP epoxygenases that metabolize arachidonic acid to produce epoxyeicosatrienoic acids(EETs).EETs exert pleiotropic protective effects,such as anti-inflammation and anti-apoptosis,on vascular damages.It is unclear whether CYP2J2 and EETs have anti-senescence effects on vascular endothelial cells.Objectives(1)To investigate whether CYP2J2 protect against retinal I/R injury induced RGC apoptosis and vascular damages.(2)To investigate the mechanisms of retinal I/R injury induced vascular damages and RGC apoptosis.(3)To investigate the molecular mechanisms of CYP2J2's protective effects on retinal vasculature.MethodsIn vivo study(1)Wild-type(WT)rats and transgenic(TG)rats specifically overexpressing CYP2J2 in endothelial cells were used for experiments.The used rats were about 8-week-old and 200 g weight.These rats were divided in 3 groups: WT,WT+I/R,TG+I/R.A rat model of acute ocular hypertension was established on WT+I/R and TG+I/R groups,while WT group acted as control.The expression levels of p53,p16,matrix metalloproteinase-9(MMP-9)and brain-derived neurotrophic factor(BDNF)were examined by immunofluorescence staining.Apoptosis levels of RGCs and capillary cells were evaluated by TUNEL staining.Vascular degeneration and senescence were tested by PAS staining and ?-galactosidase assay,respectively.(2)Wild-type rats were randomly divided in 3 groups: Ctrl,I/R,I/R+SNP.SNP,namely sodium nitroprusside,is an anti-senescence drug administered intraperitoneally before and after retinal I/R.Acute ocular hypertension was induced in groups of I/R and I/R+SNP.In the meanwhile,the I/R+SNP group received SNP before and after ocular hypertension as an anti-senescence therapy.Immunofluorescence staining was used to examine expression levels of senescence-associated proteins in situ.TUNEL assay and IB4 staining were used to evaluate apoptosis levels and vessel density,respectively.In vitro studySenescence of human umbilical vein endothelial cells(HUVECs)was induced by hydrogen peroxide(H2O2)and CYP2J2 gene was transferred to HUVECs by adenovirus.HUVECs were divided into 4 groups: Ctrl,H2O2,H2O2+Ad-GFP,H2O2+Ad-CYP2J2.The expression levels of senescence-associated proteins were evaluated by western blotting.The stage of senescence and the ability of angiogenesis were examined by ?-galactosidase staining and tube formation assay,respectively.To further investigate the molecular mechanism of senescence-associated proteins modulation by CYP2J2,we used real-time PCR to detect expression levels of several micro RNAs in the 4 groups.Moreover,we separately overexpressed and inhibited micro RNA-128-3p in HEK293 T cells and observe the resulting changes of senescence-associated protein levels using western blotting.ResultsCompared to the WT group,retinas in the WT+I/R group had increased apoptosis rates of RGCs and capillary cells as well as decreased pericytes and vessel density.In addition,the WT+I/R group had higher expression levels of p53,p16 and MMP-9,lower expression level of BDNF,as well as increased ?-galactosidase positive-stained endothelial cells.Compared to the WT+I/R group,the TG+I/R group had decreased apoptosis rates of RGCs and capillary cells as well as increased vessel density and pericytes.The TG+I/R group showed downregulated p53,p16 and MMP-9 levels.In the meanwhile,the BDNF level of the TG+I/R group was upregulated.Besides,the number of ?-galactosidase positive staining endothelial cells was smaller in the TG+I/R group.These results showed that endothelium-specific CYP2J2 overexpression reduced RGC apoptosis,endothelial cell senescence and vascular damages induced by retinal I/R injury.In comparison with the I/R group,the expression levels of p53 and p16 in I/R+SNP group had decreased.The I/R+SNP group also had increased vascular density and decreased apoptosis rates of RGCs and capillary cells.The results above suggested that the anti-senescence drug SNP attenuated endothelial cell senescence induced by I/R injury,resulting in less vascular damages and,furthermore,decreased RGCs apoptosis.The H₂O₂-treated HUVECs had upregulated expression levels of p53,p16 and ?-galactosidase than the control group.Overexpression of CYP2J2 attenuated the upregulation of p53,p16 and ?-galactosidase.The expression levels of several mi RNAs were detected by real-time PCR in groups of Ctrl,H2O2,H2O2+Ad-GFP,H2O2+AdCYP2J2.The result showed that the mi R-128-3p level was upregulated after H2O2 treatment,and the upregulation effect was abrogated in the H2O2+Ad-CYP2J2 group.We further overexpress mi R-128-3p in HEK293 T cells,as a result,levels of p53 and p16 were raised.In contrast,inhibiting mi R-128-3p in HEK293 T cells lead to suppressed p53 and p16 levels.The results of in vitro study indicated that CYP2J2 reduces endothelial cell senescence via inhibiting mi R-128-3p.ConclusionThe retinal I/R injury triggered retinal vascular endothelial cell senescence.The senescent endothelial cells resulted in retinal vascular damages,which further exacerbated RGC apoptosis.CYP2J2 attenuated vascular endothelial cell senescence and hence alleviated vascular damages.As a result,RGC apoptosis was reduced.