) The broad long term objective of this collaborative research plan is to identify new strategies for controlling the retinal neovascularization (NV) that leads to blindness in patients with diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Vascular endothelial growth factor (VEGF), a potent hypoxia-inducible angiogenic cytokine, has been implicated causally in retinal NV suggesting that VEGF and the mechanisms by which VEGF promotes angiogenesis are important targets for therapy. VEGF markedly induces expression of the alpha-1-beta-1, alpha-2-beta-1, and alpha- 5-beta-3 cell surface integrins in dermal microvascular endothelial cells (ECs), and antagonism of alpha-1-beta-1, alpha-2-beta-1, and alpha-5 integrins inhibits VEGF-driven angiogenesis in skin. In addition, targeting of alpha-5 integrins has shown promise for inhibition of retinal NV. However, the significance of alpha-1-beta-1, alpha-2-beta-1, and other beta-1 integrins for retinal NV has not been investigated. Therefore, the proposed experimental plan will: (1) investigate VEGF regulation of integrin expression by retinal ECs in vitro with particular emphasis on beta-1 integrins, and determine if the same integrin mRNAs induced by VEGF in vitro are induced similarly during retinal NV in vivo; and (2) investigate inhibition of retinal NV in vivo by monoclonal antibodies (Abs) that block alpha-1-beta-1 and alpha-2-beta-1 integrins and monoclonal Abs that block other beta-1 integrins induced in retinal ECs by VEGF. Northern analyses will test whether and to what extent VEGF, similar to its action towards dermal microvascular ECs, induces isolated bovine retinal ECs to express mRNAs encoding the alpha-1 and alpha-2 integrin subunits. Also, cell surface labeling and immunoprecipitation will be employed to determine if VEGF induces retinal ECs to express the alpha-1-beta-1 and alpha- 2-beta-1 heterodimers at the cell surface. Similarly, VEGF regulation of other beta-1 integrins in retinal ECs will also be investigated. In situ hybridization analyses of a well-defined mouse model of proliferative refinopathy will determine whether integrin subunit mRNAs induced by VEGF in vitro are similarly induced in ECs during retinal NV in vivo. Finally, specific monoclonal Abs which independently block alpha-1-beta-1, alpha-1- beta-1, and other beta-1 integrins induced by VEGF in retinal ECs will be tested for inhibition of retinal NV in vivo. Thus far, the importance of beta-1 integrins for proliferative retinopathies has not been explored. The proposed experiments will fill major gaps in knowledge by providing crucial information on the significance of beta-1 integrins for retinal NV. Moreover, it is anticipated that findings here will suggest new strategies for therapeutic intervention.
