Ras-related C3 botulinum toxin substrate 1 (Rac1) is an intracellular Rho GTPase that acts as a biologic switch in response to external stimuli. In studies testing the effects of age-related macular degeneration (AMD)-related stresses, activation of Rac1 was found to be necessary for choroidal endothelial cell invasion into the neural retina to form vision-threatening macular neovascularization. This entry summarizes the regulators of Rac1 activation, effectors of active Rac1 in choroidal endothelial cells, and mechanisms by which active Rap1, a Ras-like GTPase, may prevent active Rac1-mediated choroidal endothelial cell migration.
Rac1 is a Rho GTPases that cycles from an inactive GDP-bound form (Rac1GDP) to an active GTP-bound form (Rac1GTP) to transduce signaling in choroidal endothelial cells in response to different AMD-associated stresses (see Section 1). The biologic switch is primarily regulated by Rho guanine nucleotide exchange factors (Rho GEFs), Rho GTPase activating proteins (Rho GAPs), and Rho GDP dissociation inhibitors (Rho GDIs) (Figure 2). However, current approaches to prevent Rac1 activation by targeting these primary regulators are inefficient . Therefore, identifying alternative ways to regulate pathologic Rac1 activation is necessary.
Once activated, Rac1 interacts with several effectors in choroidal endothelial cells to elicit signaling events that lead to migration. This entry will discuss two proteins that interact with active Rac1 in choroidal endothelial cells: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and IQ motif containing GTPase activating protein 1 (IQGAP1).
NOX1 and NOX2, isoforms of the NOX family, contain active Rac1 as a subunit. In choroidal endothelial cells, VEGF-induced reactive oxygen species (ROS) generation was significantly reduced in cells transfected with Rac1 siRNA compared to control siRNA . Pretreatment with diphenyleneiodonium, a NOX inhibitor, reduced VEGF-induced choroidal endothelial cell migration , and pretreatment with apocynin, a ROS quencher, reduced choroidal endothelial cell migration in response to VEGF  or TNFα . These findings suggest that active Rac1 is an important subunit in NOX-generated ROS, which is required for choroidal endothelial cell migration in response to different AMD-related stresses. These results were corroborated in vivo using the murine laser-induced choroidal neovascularization (CNV) model, which recapitulates aspects of MNV observed in humans. C57/Bl6J mice treated with intravitreal apocynin compared to vehicle control had significantly reduced laser-induced CNV volume . Also, laser-induced CNV volume was reduced in p47phox knockout (Ncf1−/−) mice compared to littermate wild-type mice . Overall, the data suggest that NOX-mediated ROS generation is necessary for active Rac1-mediated choroidal endothelial cell migration and MNV by AMD-related stresses.
Another protein that interacts with active Rac1 in choroidal endothelial cells is IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1 was identified in immunolabeled paraffin-embedded sections of human donor eyes at the region of neovascular lesions. Knockdown of IQGAP1 by siRNA reduced Rac1 activation and migration induced by VEGF. In the laser-induced CNV model, Iqgap1−/− mice had significantly reduced experimental CNV and reduced immunofluorescent of Rac1GTP within lectin-stained sections of CNV lesions compared to littermate Iqgap1+/+ mice . Specifically, endothelial IQGAP1 knockout in mice by Cre recombinase-mediated recombination significantly reduced laser-induced CNV and immunofluorescent labeling of Rac1GTP within lectin-stained sections of CNV lesions compared to tamoxifen-treated littermate control mice that lacked Cre recombinase . Taken together, the data suggest that IQGAP1 is necessary for Rac1 activation by AMD-associated stresses. On a molecular level, previous studies have found that the GAP-related domain (GRD) of IQGAP1 binds to active Rac1 and maintains Rac1 in its active state instead of accelerating the inactivation of Rac1 . In support of this notion, choroidal endothelial cells transfected with an IQGAP1 construct that interfered with Rac1GTP binding IQGAP1 (IQ-MK24) had reduced Rac1 activation sustained by VEGF compared to choroidal endothelial cells transfected with control full-length IQGAP1 construct (IQ-WT) . Overall, the data suggest that interfering with Rac1GTP binding to IQGAP1 might reduce pathologic choroidal endothelial cell activation, migration, and the development of vision-threatening MNV.
Rap1 is a small, Ras-like GTPase that acts as a biologic switch similar to Rac1. Treatment with a pharmacologic activator of a Rap1 GEF, 8CPT-2’OME-cAMP (8CPT), significantly reduced choroidal endothelial cell transmigration across an RPE monolayer in the coculture assay, and intravitreal 8CPT administration reduced laser-induced CNV volume in the rodent laser-induced CNV models . In cultured choroidal endothelial cells, 8CPT also increased Rap1 activation and reduced Rac1 activation by VEGF  or TNFα . Furthermore, choroidal endothelial cells transduced with adenoviral vectors that expressed constitutively active Rap1a had significantly reduced VEGF-induced Rac1 activation, migration, and tube formation compared to choroidal endothelial cells transduced with control adenoviral vectors . Furthermore, adenoviral transduction of active Rap1a significantly reduced Rac1 activation and ROS generation, measured by DCFDA fluorescence, induced by TNFα . Taken together, the data suggest that activation of Rap1 antagonizes active Rac1-mediated choroidal endothelial cell migration and the development of MNV.
On a molecular level, studies have reported that active Rap1a binds to the IQ domain of IQGAP1  and displaces effectors that bind to the other domains of IQGAP1 . In support of this notion, choroidal endothelial cells transfected with a mutant construct with point mutations in the IQ domain that enhanced Rap1 interactions with IQGAP1 (IQ-3,4R)  had decreased Rac1 activation and active Rac1 binding to IQGAP1 induced by VEGF compared to choroidal endothelial cells transfected with control IQ-WT . This finding suggested that active Rap1 antagonizes Rac1 activation by also interfering with Rac1GTP binding IQGAP1, a necessary step for sustained Rac1 activation (Figure 3).