Study Of The Therapeutic Effects Of RAAV-HGFK1 On The Retinal Neovascularization

Objective:Ocular neovascularization(NV)occurs in proliferative diabetic retinopathy(PDR),age-related macular degeneration(AMD)and retinopathy of prematurity(ROP),which is the leading cause of blindness in infants,individuals of working age and the elderly in developed countries,as well as in China.Unfortunately,the number of patients who are losing their vision due to NV is increasing despite the technological advancements due to the further tissue injury and the recurrence of symptoms.Therefore,the development of a novel therapeutic approach to prevent NV progression has a vital significance.Angiogenesis is a complex multi-step pathogenetic process that involves several factors.Many growth factors have been found to play important roles in regulating neovascularization and angiogenesis,exemplified by VEGF.Hepatocyte growth factor(HGF)is an endothelium specific growth factor,which regulates cell growth and cell migration through the c-Met tyrosine kinase receptor expressed in retinal vascular endothelial cells.Recent studies have demonstrated that both serum and vitreous concentrations of HGF are significantly elevated in diabetic patients with PDR.It seems logical that HGF might be a potential mediator of retinal neovascularization,as evidenced by its mitogenic and motogenic effects on retinal endothelial cells in culture.Several HGF variants have been constructed and have been shown to have antiangiogenic activity,such as NK4(composed of the NH2-terminal hairpin domain and four kringle domains in the ?-chain of HGF),kringle 1–4,the N-terminal domain,and kringle 1(HGFK1).It has been reported that the HGFK1 could inhibit the proliferation and neovascularization of both tumor cells and microvascular endothelial cells through EGF and b FGF signaling in vitro or in a rat tumor model.The mechanisms and factors involved in neovascularization in retina are similar to those in tumor,therefore,HGFK1 probably have potential to treat retinal angiogenesis.Gene therapy refers to a technique for correcting defective genes by transducing the normal genes,recombinant genes or RNA into the individual's cells,achieves the therapeutic purposes.Gene therapy in retina has many advantages and have made significant progress.Of the recombinant viruses used in ocular gene therapy,the safety and efficacy of AAV-mediated retinal gene transfer has been demonstrated in various retinal diseases.AAV is able to transduce and mediate long-term and stable production and release of the angiogenic factors in the eye,which might be optimal for the treatment of chronic diseases associated with pathogenic angiogenesis that require long-term suppression of the angiogenic pathways.Recently,AAV-mediated RPE65 gene therapy can lead to modest improvements in visual function in Leber's congenital amaurosis patients,which further suggesting the safety and efficacy of AAV-mediated therapy.Therefore,a recombinant adenoassociated virus carrying HGFK1 might be a new effective method for treatment of NV.In this study,we aimed to evaluate the the antiangiogenic activity of rAAV-HGFK1 using the cultured bovine retinal endothelial cells and the mice model of oxygen-induced retinopathy.Methods: 1.The effects of rAAV-HGFK1 on BREC cells.1)Isolation and culture of BREC cells.Retinal microvessels isolated from fresh bovine eyes were digested by collagenase.After filtration onto nylon mesh,the cells are seeded in fibronectin coated dishes.Cultures are monitored every day and medium is changed if necessary.The endothelial cells are associated by different digestion and subculture and could be stably passaged.The endothelial origin of these cells was confirmed by the presence of the Von Willebrand factor antigen.2)BREC cells were used for in vitro studies including the proliferation and apoptosis assays.BREC cells are treated by various reagents for different time points.The effect of rAAV-HGFK1 on a vascular endothelial growth factor(VEGF)-stimulated cell proliferation were investigated by MTS assay.The cells are labelled by PE Annexin V and 7-AAD for apoptosis detection through flow cytometric analysis.3)The cells in different groups are harvested at each time points.The total protein extracts are subjected to western blot analysis for detection of expression levels of different signaling proteins.2.The effect of rAAV-HGFK1 on a mice model of oxygen-induced retinopathy(OIR).1)Animal model of proliferative retinopathy.On postnatal day 7(P7),litters of C57BL/6J mouse pups with their mothers are exposed to 75%±2% oxygen(hyperoxia)for 5 days and then returned to room air at P12.Mice of the same age are kept in room air and used as normal control subjects.At P19,retinal neovascularization are observed by pathological methods and fluorescein fundus angiography.A 0.5 ?l of rAAV-EGFP is administered by intravitreous injection in left eyes and 0.5 ?l of PBS in the right eyes as controls.The expression of EGFP is detected at P13,P17 and P21,respectively.2)Three litter containing 12 pups are used to detect the effect of rAAV-HGFK1 on OIR mice.At P3,a 0.5 ?l intravitreous injection of rAAV-HGFK1 is performed in the left eyes and 0.5 ?l of rAAV-EGFP in the right eyes as controls.Pups are then returned to their mothers and treated for establishment of OIR as previously described.At P19,the pups are killed and their eyes are enucleated and fixed 4% paraformaldehyde for 24 h,and are embedded in paraffin.Serial sections(10?m)of whole eyes were cut sagittally,through the cornea and parallel to the optic nerve,and stained with hematoxylin and eosin or performed immunohistochemical staining.Three pups from different litters were deeply anesthetized intraperitoneally and then perfused through the left ventricle with fluorescein isothiocyanate dextran for fluorescein fundus angiography.Results: 1.rAAV-HGFK1 effectively inhibited VEGF-stimulated BREC cell proliferation,rAAV-HGFK1 was also able to induce apoptosis of BREC cells.2.rAAV-HGFK1 treatment produces sustained high-level HGFK1 expression and acts through inhibiting ERK activation as well as promoting p38 MAPK activation.3.The fluorescein fundus angiography of OIR mice 19 days after born(P19)display vessel exudation and significant reduce of retinal microvessel perfusion.In serial paraffin cross-sections,the endothelial cell nuclei break through internal limiting membrane and enter into the vitreous body,some form the vessel cavity.Green fluorescein protein were detected in P17 mice retina after intravitreal injection of rAAV-EGFP,and enhanced in P21.4.the P19 mice received rAAV-HGFK1 injection had an evident improvement of retinal ischemia and microangiopathy,moreover,the quantification of proliferative retinopathy show less neovascular endothelial nuclei compared with contral group.The CD31 and VEGF in rAAV-HGFK1 injected group were found less than control group.Conclusions:rAAV-HGFK1 was shown to inhibit vascular endothelial growth factor(VEGF)-stimulated cell proliferation and promote the cell apoptosis in vitro,which regulate the retinal angiogenesis probably through the MARK pathway including ERK and p38 MAPK signaling factors.It also had anti-neovascularization activity in the retinal neovascularization of a mouse OIR model.rAAV-HGFK1 may lead to new potential drug discoveries and the development of new treatments for pathological retinal angiogenesis.