Protective Effect Of Adenovirus-mediated Pigment Epithelium-derived Factor On Retinal Ischemia-Reperfusion In Rats

ObjectiveRetinal ischemia is a serious and common problem that occurs as a result of acute vascular occlusion and leads to loss of vision in a number of ocular diseases such as acute glaucoma,diabetic retinopathy,hypertensive retinopathy,and retinal vascular occlusion.Neurotrophic and growth factors,such as ciliary neuro trophic factor(CNTF),basic fibroblast growth factor(bFGF),nerve growth factor(NGF),brain - derived neurotrophic factor(BDNF),pigment epithelium derived factor(PEDF),and hepatocyte growth factor(HGF)have demonstrated neuroprotective effects against retinal ischemia -reperfusion injury.Pigment epithelium - derived factor(PEDF),a 50 - kDa protein is secreted by human fetal retinal pigment epithelial cells and has been shown to induce neuronal differentiation of human Y-79 retinoblastoma cells in vitro.PEDF is potentially both a promising endogenous inhibitor of angiogenesis and a neuroprotective protein. PEDF has been shown to be protective in models of inherited photoreceptor degeneration,hydrogen peroxide induced neuronal cell death,ischemic retinal injury,and retinal light damage.Because of the short halflives of PEDF,repeated intravitreal injections would be necessary.This would likely cause many undesirable side effects.To overcome the damage induced by repeated injections, Viral vectoring of genes into the ocular tissues provides for sustained local delivery of therapeutic agents.The eye,being both small and a relatively isolated compartment,requires a comparatively small amount of vector to transfect a large number of ocular cells.viral vectors have been proven to be useful in transfecting ocular cells.In fact,neurotrophic factors could be delivered to the retina using gene therapy.This study is intended to examine the protective effects and mechanism of adenovirally delivered pigment epithelium derived factor(Ad -PEDF)on experimental ret inal ischemia reperfused injury(RIR).Methods1.Construction of Aecombinant Human pigment epithelium derived factor by the homologous recombination in bacterial.The gene of PEDF was digested from pcDNA3.1 - PEDF,PEDF cDNA were cloned into adenovirus shutle vector pSu - CMV by standard procedure.The recombinant adenoviral plasmid pSu -CMVPEDF was identified and the correct clones containing target gene in right direction were selected,each bearing a loxP site and the latter containing a cre site,were cotransferred into the adenoviral packaging 293 cell lipofectamine mediated gene transfer method to recombinant and pack the virus.After identified, the desired Ad vectors were purified by cesium chloride density gradient ultra centrifuge and titrated.2.Wistar rats weighing 180-220 g were used in all subsequent experiments. RIR was induced in 96 adult wistar rats by increasing intraocular pressure via an intracameral infusion.The animals were prepared and anesthetized, the anterior chambers of both eyes were cannulated with a gauge infusion needle connected to a reservoir containing a balanced salt solution,The intraocular pressure was raised to 110 mmHg by elevating the reservoir.The infusion needle was removed from the anterior chamber 60 min later.Retinal ischemia and reperfusion were confirmed by the whitening of the fundus and restoration of the retinal blood flow.3.All of the rats were divided into four groups:normal control group;RIR group;RIR + Ad - PEDF treatment group and RIR + Ad - CMV control group. The groups were subdivided into groups:12hours,24hours,72 hours and 168 hours after reperfusion randomly,in Ad- PEDF treatment group,Gene delivery to retinal cells was achieved through intravitreal injections of recombinant adenoassociated virus expressing PEDF(Ad - PEDF)1μL(3.8×10~9 pfu/ml)after retinal ischemia.Ad - CMV treatment group was injected correspondingly. 4.The synthesis and accumulation of PEDF within the retina were determined using western blot after after reperfusion 12,24,72,168hour.The neuroprotective effects of Ad-PEDF were evaluated by determining the preservation of the inner retina thickness and the cell counts in the retinal ganglion cell layer by HE staining method after reperfusion 12,24,72,168hour,Ischemic changes were evaluated by measuring the inner retinal thickness,which was defined as the thickness between the inner limiting membrane and the boundary of the outer nuclear layer and the outer plexiform layer.Measurements were taken at intervals of 100μm in a range of retina,The numbers of RGC were taken at intervals of 100μm in a range of retina at 400 magnification.The levels of RGC apoptosis were measured using the TdT- dUTP terminal nick- end labeling(TUNEL) method 12,24,72 hour after reperfusion.Eletroretinograms(ERGs)were performed to determine the functionality of the retinas.Flash ERG results were recorded to assess the retinal function of Ad - PEDF injected eyes and control eyes.ERG studies were performed in both eyes before ischemia(baseline), 12h,24h,72h and 168h after reperfusion.The amplitudes of the b-waves and Ops wave were observed.Results1.The linealinzed pSuCMVPEDF was transformed into 293cell.There were positive recombinant plasmids.PCR test and gene sequencing indicated that the recombinant adenovirus plasmid pSuCMVPEDF contained PEDF gene.Titer reached 3.8 10~9pfu/ml after density gradient ultracentrifuge.2.Gene expression of PEDF was demonstrated through western blot,Quantitative analysis showed that PEDF expression was higher in Ad -PEDF groups than in control group at every time point.3.Significant ameliorative effect on the thickness of the inner retina was observed in Ad - PEDF treated eyes.There was also significantdifference between Ad - PEDF treated eyes and untreated eyes.The loss of cells in RGC layer was apparent after ischemic treatment,more cells in the RCG layer were retained in theAd - PEDF treated eyes than in the Ad - CMV treated eyes or in the untreat- ed eyes.These results were statistically significant(p＜0.05 respectively). More TUNEL positive RGCs were noted in Ad- CMV injected eyes and untreated retina,while only sparse TUNEL positive RGCs were noted in Ad - PEDF injected eyes.there were statistically fewer apoptotic cells in Ad-PEDFinjected eyes than inAd-CMV injected eyes or untreated eyes.Ad-PEDF treated eyes had statistically fewer apoptotic cells in the RGC layer than the control eyes(p＜0.05 respectively).4.Began from the 12hours after reperfusion,in Ad- CMVcontrol group,the amplitudes of ERG b - wave and Ops wave reduced continuously,significantly larger amplitudes of ERG b - waves and Ops wave were measured in Ad - PEDF treated eyes than those in Ad- CMV treated eyes or control eyes.Conclusions1.The efficiency and reliability of this Cre- mediated site-specific recombination method is a convenint and efficient method to prepare recombinant adenovirus plasmid PEDF,This offers a good gene transfer vector for the gene therapy。Efficient and stable transfer of PEDF gene could be achieved by adenovirus delivery into the retina of rats.2.Intravitreal injections ofAd -PEDF resulted in PEDF protein expression. in the retina.3.Intravitreal injection of Ad - PEDF moderately protected the inner retina and the cells in the RGC layer of eyes after ischemia reperfusion injury.4.Intravitreal injection of Ad-PEDF moderately protected rat retina from ischemia-reperfusion injury,possibly by preventing apoptosis in retinal cells.5.ERG analyses confirmed moderate protection of the retina from ischemia reperfusion injury.6 Our results demonstrate the possibility of using gene therapy of PEDF for treating ischemic retinal injuries.