The pathophysiology of neovascular age-related macular degeneration (nvAMD) is complex and involves the impact of genetic predisposition, environmental stresses and advanced aging on signaling events that overwhelm retinal/choroidal homeostasis and enable activation and migration of Choroidal Endothelial Cells (CECs) across the Retinal Pigment Epithelial (RPE) cell monolayer into neural retina to become Choroidal Neovascularization (CNV). Using physiologically relevant human heterotypic cocultures to model RPE/CEC interactions, we now focus on mechanisms necessary for CEC transmigration of the RPE, including crosstalk among activated signaling pathways in CECs that involve angiogenic (VEGF and CCR3), inflammatory (TNF?), and oxidative factors that feed-forward to induce Rac1-dependent CEC migration. In particular, our findings -- that (i) the scaffolding protein IQGAP1 that brings together multiple signaling cascades to enable biologic events to occur, is necessary for CEC transmigration, together with (ii) Thy-1 is overexpressed in CECs from older eyes and eyes exposed to AMD-related factors -- support the following hypothetical framework that will be tested in the next funding period: that (1) complex intracellular signaling cascades may be linked together to a) activate CEC migration through the IQGAP1 GRD domain, to enable Rac1-mediated CEC activation and b) enable CEC migration via ECm-induced Thy-1/IQGAP1 interactions with ECm, and that 2) IQGAP1-facilitated pathologic signaling might be inhibited when activated Rap1a binds to the IQGAP1, thus restoring CEC quiescence and preventing CNV.
Specific Aim 1 is to test the prediction that Rac1/IQGAP1 binding affects CEC activation and migration induced by AMD-related ligands involving angiogenesis, inflammation and oxidation.
Specific Aim 2 is to test mechanistic roles of Rap1/IQGAP1 binding on CECs and CNV formation.
Specific Aim 3 is to test mechanisms underlying Thy-1/IQGAP1 interactions in activating CECs and enabling CNV formation. Tools include isolated human CECs; mutant constructs to different IQGAP1 domains that inhibit direct binding of the GTPases Rac1 or isoforms of Rap1 (to test predictions regarding induced CEC migration); engineered adenoviral constructs (to introduce constitutively active, dominant-negative or wild type Rap1 isoforms or Rac1), or microRNAs (to test mechanisms of action); pharmacologic agents that activate Rap1; and knockout mice to Rap1 isoforms and IQGAP1 (to test mechanisms involved in laser-induced CNV with the Micron IV); These studies will test whether Rap1 binding to IQGAP1 inhibits steps necessary for CNV formation and restores CECs to a quiescent state. Results will inform future research on therapies to target multiple causal events in CEC activation and migration in nvAMD and restore CEC quiescence while reducing risks of current angiogenic inhibitor treatments. !
Neovascular age-related macular degeneration is a leading cause of blindness worldwide. Current treatments involve methods to inhibit angiogenesis, which could lead to nutritional deprivation and hypoxia to the outer retina and over time adversely affect vision. We use novel approaches to regulate signaling pathways in order to prevent the invasion of blood vessels into the neural retina and safely but effectively reduce vision loss from age-related macular degeneration.
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