Experimental Study On Inhibition Of Retinal Neovascularisation By Gene Transfer Of Extracellular 1-3 Domain Of VEGF Receptor KDR

Retinal neovascular disorders, such as diabetic retinopathy and retinopathy of prematurity, are the principal causes of blindness. The potent treatment is the inhibition of retinal angiogenesis. Current treatments including laser photocoagulation and vitrectomy are of limited efficacy and cause significant adverse effects sometimes. Over the past several years, a variety of proteins have been identified as potential targets of anti-angiogenesis therapy, but poor results have been given in some clinical trails of the antiangiogenic strategies.Vascular endothelial growth factor (VEGF) is a potent endothelial cell specific mitogen that plays a critical role in angiogenesis. VEGF exhibits high affinity binding to two distinct endothelial cell receptor tyrosine kinases, the fms-like tyrosine kinase Flt-1 and the kinase insert domain containing receptor KDR. Recent findings suggested that KDR but not Flt-1 was able to mediate the mitogenic and chemotactic effects of VEGF. Blocking the VEGF- KDR pathway may inhibit angiogenesis. KDR belongs to receptor tyrosine kinases (RTKs) family and was expressed almost exclusively in endothelial cells. KDR has a typical tyrosine kinase receptor structure with 7 immunoglobulin (Ig)-like domains in the extracellular region, as well as a long kinase insert in the tyrosine kinase domain. Evidence shows that IgG-like domains 2 are sufficient for tight binding, domain 1-3 is necessary for tight binding and domains 4-7 are not essential for signaling.In this study, we first constructed expression vectors of enhanced green fluorescent protein gene and extracellular 1-3domain of KDR:KDRn3, and second trasfected recombinant plasmid pEGFP-N1/ KDRn3 to human umbilical vein endothelial cells (ECV-304) by cationic liposome mediating to evaluate the effect of cell proliferation in vitro, and then we observed the expression and location of the pEGFP-N1/ KDRn3 in retina by trasfectting the plasmid to the retina of C57BL/6J mouse, finally we investigated the effect of gene transfer of pEGFP-N1/ KDRn3 on the eyes of C57BL/6J mice of ischaemia-induced retinal neovascularisation.Materials and Methods:1.The KDRn3 gene was amplified by PCR with the recombinant plasmid pcDNA3.1/ KDRn3 as the template and the fragment was cloned into pMD18-T vector. KDRn3 gene was released from pMD18-T KDRn3 and then inserted to plasmid pEGFP-Nl with double enzymatic digestion.2. Human umbilical vein endothelial cells (ECV-304) were cultured and then trasfected by liposomes mediated pEGFP-N1/ KDRn3.The expression of EGFP was observed under fluorescence microscope, and the KDRn3 protein expression was tested with immunohistochemistry. The effect of cell proliferation was determined by methyl thiazolyl tetrazolium(MTT) method. The effect on cell cycle was examined by flow cytometric analysis.The culture supernatant was identified by ELISA.3.20 C57BL/6J mice were randomly divided into 5groups,four groups were intravitreal injected with the optimal liposome plasmid mixture,others was control group. 2d,5d,7d,14d after injection, the pEGFP-N1/ KDRn3 expression was observed by using fluorescence microscopy, and morphological changes were examined by using paraffin embedded sections, HE staining.4. mice were exposed to 75±2 % oxygen for 5 days, then returned to the room air to induce retinal neovascularization. The optimal liposome plasmid mixture was injected into the vitreous . We evaluated retinal neovascularisation by counting the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina and measuring the areas of non -perfusionsin in central retina.Results:1. The eukaryotic expression vector pEGFP-N1/KDRn3 was successfully constructed. DNA sequencing and blast search showed that KDRn3 completely matched with the DNA sequence in GenBank.2. KDRn3 protein was expressed in ECV-304 cells and secreted in the culture supernatant. With the time prolonging,they became stronger and stronger.The proliferation of ECV-304 was inhibited by KDRn3 transfection with the ratio of the S phase decreased.3. pEGFP-N1/KDRn3 protein was expressed in the retinal ganglion layer 2d after injection. After 5d or 7d, it was expressed both in the ganglion layer and in the inner layer. After 14 days, it showed the weakened expression. Araffin embedded sections, HE staining showed that there was no sifnificant difference in morphologist between control group and pEGFP-N1/KDRn3 injecting group.4.Retinal wholemount preparation of retinal neovascular animal model showed that prominent neovascular tuft and fluorescein leakage and large areas of non -perfusionsin in central retina.Fewer neovascular tufts and fewer areas of non-perfusionsin could be seen after pEGFP-N1/KDRn3 injection. There were statistic differences between control group and pEGFP-N1/KDRn3 injecting group with the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina and the areas of non -perfusionsin in central retina.Conclusion:1. The successful construction of the eukaryotic expression vector pEGFP-N1/KDRn3 has been achieved.2. pEGFP-N1/KDRn3 can be transferred to endothelial cells by cationic liposome mediating and inhibit the proliferation of endothelium cells.3. Cationic liposome can mediate pEGFP-N1/KDRn3 gene into the retina of the C57BL/6J mice effectively,4. pEGFP-N1/KDRn3 gene transfer can effectively inhibit retinal neovascularisation in C57BL/6J mice of ischaemia-induced retinal neovascularisation.Discussion:VEGF plays a critical role in angiogenesis. KDR, which is high-affinity receptor tyrosine kinases of VEGF leads to endothelial cell proliferation, migration, and increased vasopermeability. so KDR is a good target to treating retinal neovascular disorders. KDRn3, a soluble forms of KDR, is the extracellular 1-3domain. The angiostatic activity of KDRn3 results from inhibition of VEGF by two mechanisms. It causes both sequestration of VEGF, to which it binds with high affinity, and forms inactive heterodimers with membrane-spanning isoforms of the VEGF receptors Flt-1 and KDR. A recent study shows Flk-1 DNA vaccine produced by cloning extracellular domains 1-3 of flk-1 could significantly dimish microvessel density (MVD) of the tumor. Our results have shown the proliferation of ECV-304 was inhibited by KDRn3 transfection in vitro and effectively inhibit retinal neovascularisation in C57BL/6J mice of ischaemia-induced retinal neovascularisation in vivo.Human eye is such kind of organ that shows particular potential for gene therapy. Intraocular gene delivery is experimentally easy and results in minimal spreading of the vectors and negligible leakage of the therapeuticfactor to extraocular organs. High intraocular concentrations of the therapeutic agent can be achieved by using amounts of vector much lower than those necessary for systemic gene delivery. Finally, the immuno-privileged status of the eye may reduce the potential immune response against the vectors and facilitate long-term transgene expression. Comparing with the systemic administration of angiostatic proteins, there are many advantages of a local gene transfer approach to retina. Although potentially efficacious, the systemic administration of angiostatic proteins risks important adverse systemic effects. In addition to these concerns, the production of recombinant proteins is technically difficult to gain and their manufacture could be more expensive. The local delivery of angiostatic agents offers significant potential advantages. The relatively short half-life of proteins delivered by intravitreal injection is such a thing that frequently repeated administration would be necessary to maintain therapeutic levels and would pose a high cumulative risk of local complications, including intraocular infection, vitreous haemorrhage and retinal detachment. .In this study , we have showed that cationic liposome can mediate pEGFP-N1/ KDRn3 gene into the retina of the C57BL/6J mice effectively.In conclusion, gene transfer of KDRn3 consistently controls angiogenesis in experimental retinal neovascularisation. Our findings demonstrate the feasibility of an gene therapy in vivo approach to the clinical management of angiogenesis in the retina.