Observation On Reversible Retinal Vascular Closure Resulting From VEGF Induced Leukostasis

Background: Recently,retinal vessel closure as the most consequential vascular change in diabetic retinopathy(DR)and retinal vein occlusion(RVO)resulting in retinal nonperfusion and occurrence of ischemia are the most prevalent ischemic retinopathy causing vision loss of grown-ups over world.There are several clinical trails working on the therapeutic effects of VEGF neutralizing protein on diabetic macular edema(DME)and macular edema(ME)resulting from RVO,and additional benefits are obtained that suppression of retinal VEGF could cause part of previously closed vessels re-opened and prevent the progression of vessles closure and occurrence of microanurysms,which suggests that VEGF might play a role in the vessel closure in DR or RVO.On basis of leukostasis having been reported to contribute to the retinal vessels closure at early stage of DR,we assume that VEGF might induce reversible retinal vessel closure via initiation of leukotasis and suppression of VEGF could cause reopening of these vessels,which is not reported by now.Hence this study is intended to investigate the assumption and uncover the potential machenism involved.Objects:(1)To investigate retinal leukostasis induced by VEGF in different animal models.(2)To observe whether leukostasis induced by VEGF could promote retinal vessel closure followed by retinal hypoxia.(3)To explore whether turning-off increased expression of VEGF or blocking VEGF with VEGF neutralized protein could reverse retinal vessel closure.(4)To uncover mechanism involved in VEGF induced leukostasis.Methods:(1)C57BL/6 mice were given human VEGF at different doses intravitreously(0,50,100,20,50,1000ng/?l)followed by perfusion with rhodamine or FITC labeled conconavalin(ConA)to label adherent leukoctyes in retinal vessels and counting of adherent leukocytes.(2)Tet/opsin/VEGF double transgenic mice with high expression of VEGF in retinal photoreceptors intiated by doxycycline water or injection and rhodopsin/VEGF transgenic mice in which mildly increased expression of VEGF in photoreceptors driven by rhodopsin promoter starting at P7 were perfused with rhodamine labled VEGF with leukocytes counting.(3)Sodium fluorescein angiography(FA)was performed and fundus photographs were collected showing retinal perfusion,and retina was stained by hypoxyprobe together with FITC-CoA perfusion to test retinal hypoxia related to leukotasis.(4)Turning off increased VEGF in tet/opsin/vegf mice by cessation of doxycycline injection or trapping VEGF by aflibercept known as VEGF neutralizing protein,retinal perfusion of same mouse was performed FA and counted adherent leukocytes.(5)In vitro,HRECs were stimulated by VEGF followed assay of adhesion molecules including ICAM-1,VCAM-1,selectins by RT-PCR and activation of NF-?B by transcription activity assay,MRECs were stimulated by VEGF followed by immunofluorescence staining of VCAM-1.In vivo,expressions of the above adhesion molecules in the retinas at RNA level were quantified by RT-PCR.Additionally,expression and distribution of VCAM-1 in retina were analyzed by WB and IF assay at protein levels.Translocation of NF-?B(p65)to nucleus was tested by WB,and activity of transcription was detected by subretianl transfection of NF ?B(p65)lucifersase reporter vector followed by luciferase assay.Subtypes of adherent leukocytes detected by IF with specific antibodies F4/80,Ly6 G binding to monocyte,neutrophil respectively.(5)Blockade of VCAM-1,VEGFR1 with intravenous administration anti-VCAM-1 antibody,anti-VEGFR1 antibody via tail vein injections followed by observation of leukostasis in mice injected with VEGF or tet/opsin/VEGF mice.Results:(1)In mice injected with more than 50 ng VEGF,a mass of leukocytes labled with ConA were seen 24 hours after injection.Most of adherent leukocyte plugged branches of vessels in lower dose group,while clots of leukocytes adherent to dilated veins and the downstream vessels in higher dose group.(2)Leukocytes labeled with ConA adherent to retinal vessels were dramatically increased in tet/opsin/vegf double transgenic mice,and the numbers were increasd in line with the up-regulation of VEGF.Accordingly,FA results showed retinopathy sencondary to VEGF as definitely dilated retinal veins,large area of vascular leakage,pieces of retinal vessels closure and non-perfusion at posterior polar 2,3 days after doxycycline induction.Positive hypoxyprobe staing were accumulated in retinal tissue where vessels were plugged with adherent leukocytes,which indicated that hypoxia occurred.However,in rho/vegf imce,leukostasis mostly occurred in small braches of retinal vessles,cappillaries or furcation and presented as single leukocyte or a small clot,and adherent leukocytes counting were slightly increased from 20 days after birth,reached peak at 7 months old and sustained until 15 monthes.Retinopathy of 7ms,12 ms old rho/vegf mice in FA fundus images appeaed as disturbed distribution of retinal vessels,subretinal neovascularization with less severe leakage compared to tet/opsin mice,multimple small area of retinal non-perfusion focused on posterior polar and some of them surrounding neovascularization.(3)Closed vessels were re-opened 14 days after turning-off VEGF in tet/opsin mice together with reduction of leukostasis;in rho/vegf mice treated with aflibercept,part of retinal non-perfusion reperfused and neovascualrizaiton dimished;while,tet/opsin/vegf mice injected aflibercept intravitreously reduced leukostasis significantly,and no identical retinal nonperfusion were seen in FA phothograms.(4)mRNA of VCAM-1 was up-regulated after VEGF stimulation both in vitro and in vivo,both WB and IF showed VCAM-1 was up-regulated at protein level ad located on the superficial retinal vessels,and blocking VCAM-1 partially reduced leukostasis induced by VEGF;NF-?B translated to nucleus shown by WB and more luciferase activated were tested in accordance with NF-?B transcription activity assay in vitro.(4)Monocyte are the main leukocytes adherent to vessels,while a mall portion of neutrophils were also involved shown by IF.Leukostasis was slightly reduced by blocking VEGFR1 expressed in leukocytes in mice injected with VEGF compared to PBS.Conclusions: 1)This study demonstrates that high levels of VEGF in the retina recruit leukocytes into the retinal vasculature at least in part through stimulation of VEGFR1 and promote endothelial cell adhesion and vessel plugging through activation of NF-?B and increased expression of VCAM-1.2)This study explains the clinical observation of reperfusion of previously nonperfused retinal vessels and reduced progression of nonperfusion in patients with ischemic retinopathies treated with intraocular injections of a VEGF-neutralizing protein.3)Our study also indicates that despite different insults responsible for onset of retinal ischemia in RVO,diabetic retinopathy,and other ischemic retinopathies,they evolve into very similar diseases in which VEGF is the driver of an accelerating spiral of disease progression.4)Potential insight is raised on treatments of those ischemic retinopathy that individually sustained suppression of VEGF or combination with neutralizing VEGFR1,VCAM-1 prevent retinal non-perfusion in early stage of ischemic retinopathy including RVO and DR and progression of ischemic retinopathy in future.