A DNA Vaccine Against FLK-1 Inhibits Choroidal Neovascularization In Mice

PrefaceChoroidal neovascularization ( CNV) is associated with various disorders, often causing severe loss of vision and eventually blindness. Among these disorders, age -related macular degeneration (AMD) are the most prevalent in developed countries. The current treatments of AMD include convention laser therapy and photodynamic therapy. The conventional laser therapy results in scoto-ma and significant central visual loss, whereas photodynamic therapy needs to be repeated frequently because of significant recurrence, and is reserved for a select type of subretinal neovascularization. Therefore, new therapeutic approaches are sought for diseases with CNV. Recently, the use of antiangiogenic therapies , aimed at preventing or regressing neovascularization and based on molecular insights into the pathogenesis of the disorders, have been proposed. One of these promising novel approaches is DNA vaccine.A DNA vaccine can be defined as a plasmid containing a viral, parasitic, or bacterial gene that can be expressed in mammalian cells (in the case of infectious diseases) or a gene encoding a mammalian protein (in the case of nonin-fectious diseases, such as cancer, autoimmunity, and allergy) . The plasmid DNA is delivered in vivo, resulting in antigen expression by the host's cells. Thus, the antigen may be available for both induction of antibody production and to the Class I - restricted MHC antigen presentation pathway for CTL induction. Since DNA vaccines can be engineered like other recombinant DNA, modifications to the gene can be readily made and tested for improved effectiveness.It has been widely known that vascular endothelial growth factor (VEGF) , a potent endothelial cell mitogen and angiogenic factor, is crucial for normal an-giogenesis and it also plays an important role in pathological angiogenesis, including CNV. VEGF - mediated angiogenesis is induced by binding of VEGF to its endothelial cell receptors. Among these receptors, the vascular endothelial growth factor receptor 2 (VEGFR - 2, also known as FLK - 1) that binds the five isomers of murine VEGF has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during pathological angiogenesis. Thus FLK -1 is strongly implicated as a therapeutic target for pathological angiogenesis. Recently, a FLK - 1 based DNA vaccine was reported to achieve an antitumor immune response by evoking a T cell mediated immune response a-gainst proliferating endothelial cells overexpressing this growth factor receptor in the tumor vasculature. In our study, we will examine the effect of the FLK - 1 based DNA vaccine on experiment CNV with an investigation into its possible mechanisms, and also will provide further experimental evidence supporting the possibility that DNA vaccine could have potential for the treatment of ocular neo-vascularization.Materials and Methods1. Preparation of the DNA vaccineThe auxotrophic Salmonella typhimurium ( AroA/A ~ ) was electroporated with the expression vector encoding murine FLK - 1 ( pcDN3. 1 - FLK1) and empty vector (pcDNA3. 1) , used as DNA vaccine liquor and control liquor respectively.2. Oral immunizationTwo Groups of female C57BL/6J mice were immunized by oral gavage 3 times at 2 - wk intervals with 100 ul PBS containing 1 x 108 Salmonella typhimurium (AroA/A) transformed with pcDNA3. 1 - FLK1 (FLK - 1 group) or pcDNA3.1 ( vector group). A further group not received any administration was used as a control.3. Experimental CNV modelsTwo weeks after oral immunization, experimental CNV was generated by laser - induced rupture of Brucks membrane in these mice. Production of a bub-ble at the time of laser exposure, which indicates rupture of Brucks membrane, is an important factor in inducing CNV, and therefore only mice in which a bubble was produced for all 4 bums were included in the study.4. Evaluation of anti - angiogenic effects10 days after laser treatment, the size of CNV lesions was evaluated quantitatively by one of two different techniques, measurement of the area of CNV in choroidal flat mounts or measurement of the integrated area of CNV on serial sections. The expression of FLK -1 in CNV was also assessed by immunohisto-chemical examination.5. In vivo depletion of CD8+ T cells and CD4+ T cellsTo reveal whether CD8 + T cells or CD4 + T cells are responsible for the anti - angiogenic response, depletion of CD8 * and CD4 T - cell subsets was accomplished by intraperitoneal injection ( on day 2 before laser treatment and 5 after laser treatment) of rat anti - mouse anti - CD8 mAb ( GK1. 5) and anti -CD4 (53 -6.7) respectively in FLK - 1 group. CNV was qualitatively assessed by immunolabeling on whole - mount preparations and histopathologic examination quantitatively.6. Association of cytotoxic T cells with vascular endotheliumTo reveal the localization of CD8 + T cells to their target site, we stained these cells with PE - conjugated rat anti - mouse CD8a mAb and marked endo-thelial cells with biotinylated isolectin B4 and FITC - conjugated avidin using tissue sections from the experimental models, vaccinated with pcDNA3. 1 -FLK1 or the control ones.Results1. The DNA vaccine against FLK -1 inhibits development of CNVIn the measurement of the area of CNV in choroidal flat mounts or measurement of the integrated area of CNV on serial sections 10 days after laser treatment , mice that did not receive any administration showed large areas of CNV at sites of rupture of Bruch's membrane. Mice that received vector liquor showed areas of CNV that were very similar to those untreated mice ( P > 0. 1). Mice...