Active Rap1‐mediated inhibition of choroidal neovascularization requires interactions with IQGAP1 in choroidal endothelial cells

Abstract

Neovascular age‐related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision‐threatening CNV. IQGAP1 also binds the GTPase, Rap1, which when activated reduces Rac1 activation in CECs and CNV. In this study, we tested the hypothesis that active Rap1 binding to IQGAP1 is necessary and sufficient to reduce Rac1 activation in CECs, and CNV. We found that pharmacologic activation of Rap1 or adenoviral transduction of constitutively active Rap1a reduced VEGF‐mediated Rac1 activation, migration, and tube formation in CECs. Following pharmacologic activation of Rap1, VEGF‐mediated Rac1 activation was reduced in CECs transfected with an IQGAP1 construct that increased active Rap1‐IQGAP1 binding but not in CECs transfected with an IQGAP1 construct lacking the Rap1 binding domain. Specific knockout of IQGAP1 in endothelial cells reduced laser‐induced CNV and Rac1 activation in CNV lesions, but pharmacologic activation of Rap1 did not further reduce CNV compared to littermate controls. Taken together, our findings provide evidence that active Rap1 binding to the IQ domain of IQGAP1 is sufficient to interfere with active Rac1‐mediated CEC activation and CNV formation.

Regulation of Rac1 Activation in Choroidal Endothelial Cells: Insights into Mechanisms in Age-Related Macular Degeneration

“…These studies suggest that Rap1 activation might prevent endothelial cell migration by protecting barrier integrity. In line with this notion, 8CPT treatment significantly reduced choroidal endothelial cell migration across an RPE monolayer in the transmigration assay, and intravitreal 8CPT administration reduced lectin-stained CNV volume in rodent laser-induced CNV models [26,31,88,89]. 8CPT also increased Rap1 activation and reduced Rac1 activation by VEGF [31] or TNFα [26] in cultured choroidal endothelial cells.…”

“…In line with this notion, 8CPT treatment significantly reduced choroidal endothelial cell migration across an RPE monolayer in the transmigration assay, and intravitreal 8CPT administration reduced lectin-stained CNV volume in rodent laser-induced CNV models [26,31,88,89]. 8CPT also increased Rap1 activation and reduced Rac1 activation by VEGF [31] or TNFα [26] in cultured choroidal endothelial cells. Although 8CPT is an EPAC-specific analog, EPAC can activate several downstream signaling effectors in addition to Rap1 [90].…”

“…Since Wang et al observed IQGAP1 protein expression in RPE/choroid lysates from wildtype mice, the authors bred commercially available mice to generate a tamoxifen-inducible Cre-loxP endothelial-specific IQGAP1 knockout mice along with littermate control mice that lack Cre recombinase and, therefore, continued to express endothelial IQGAP1 following tamoxifen administration. Tamoxifeninduced endothelial knockout of IQGAP1 in Cre-loxP mice resulted in significantly reduced laser-induced CNV and reduced Rac1GTP expression in cryosections of laser-induced CNV lesions when compared to tamoxifen-injected littermate controls [31]. Taken together, the in vivo data suggest that endothelial IQGAP1 is necessary for the development of CNV by interacting with active Rac1.…”

“…Although 8CPT is an EPAC-specific analog, EPAC can activate several downstream signaling effectors in addition to Rap1 [90]. Compared to adenoviral transduction with a vector that expressed GFP as control, choroidal endothelial cells transduced with adenoviral vectors that expressed GFP-tagged constitutively active Rap1a had significantly reduced Rac1 activation, migration, and tube formation induced by VEGF compared to PBS [31]. Adenoviral transduction of active Rap1a also significantly reduced choroidal endothelial cell Rac1 activation and ROS generation, measured by DCFDA fluorescence, in response to TNFα compared to PBS [26].…”

“…Inhibiting endogenous active Rac1-mediated signaling in choroidal endothelial cells by transduction with either green fluorescent protein (GFP)-tagged dominant negative Rac1 or a GFP-tagged p21-activated kinase binding domain (PBD) protein reduced migration across the RPE cell monolayer compared to choroidal endothelial cells transduced with GFP as control [25]. Studies have since demonstrated that Rac1 is activated in choroidal endothelial cells by several AMD-associated stresses, tumor necrosis factor alpha (TNFα) [26], an example of an inflammatory cytokine; vascular endothelial growth factor (VEGF) [27][28][29][30][31][32] or C-C motif chemokine 11 (CCL11) [30], angiogenic stimuli; reactive oxygen species (ROS) [26]; and 7-ketocholesterol (7KC) [29,33], an oxidized cholesterol that accumulates in human Bruch's membrane (Figure 1). A study also demonstrated CD93, a transmembrane glycoprotein [34] that is overexpressed in endothelial cells within choroidal neovascular membranes [35,36], was necessary for Rac1 activation and migration in human umbilical vein endothelial cells (HUVECs) [37].…”