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.

Keywords: IQGAP1; Rac1GTP; Rap1GTP; choroidal neovascularization; vascular endothelial growth factor.

Fig. 1. Rap1 activation reduces VEGF-induced Rac1 activation in a dose-dependent manner.

A) Representative western blot images depicting Rac1 activation by immunoprecipitation of Rac1GTP and blot of total Rac1 (row 1), total Rac1 (row 2), Rap1 activation by immunoprecipitation of Rap1GTP and blot of total Rap1 (row 3), total Rap1 (row 4), and β-actin (row 5) in lysates from CECs treated with 0.05% DMSO (0 μM 8CPT-AM), 0.25 μM, 0.5 μM, or 1 μM of 8CPT-AM in the presence of PBS or VEGF (50 ng/mL). B) Quantification of Rac1 activation (top) and Rap1 activation (bottom) by densitometry analysis (Normalized mean±SEM, *p<0.05 vs. 0 μM of PBS, ^†p<0.05 vs. 0 μM of VEGF, n=3 per group from 3 independent experiments)

Fig. 2. Rap1 activation reduces VEGF-induced Rac1 activation, migration, and tube formation by interacting with IQGAP1 in CECs.

A) Representative western blot images that show Rap1GTP interactions with IQGAP1 determined by co-immunoprecipitation (row 1), total IQGAP1 (row 2), Rap1 activation by immunoprecipitation (row 3), and total Rap1 (row 4) in lysates from CECs treated with PBS and 0.05% DMSO (Control), VEGF (50 ng/mL) and Control, PBS and 8CPT-AM (1 μM), or VEGF and 8CPT-AM for 30 minutes; B) Quantification of Rap1GTP interactions with IQGAP1 by densitometry analysis (Normalized mean±SEM, *p<0.05 vs. PBS of Control, †p<0.05 vs. VEGF of Control, n=3 per group from 3 independent experiments); C) Representative western blot images depicting Rac1GTP interactions with IQGAP1 determined by co-immunoprecipitation (row 1), total IQGAP1 (row 2), Rac1 activation by immunoprecipitation of Rac1GTP and blot of total Rac1 (row 3), and total Rac1 (row 4) in lysates from CECs treated similarly as Fig. 2A; D) Quantification of Rac1 activation by densitometry analysis (Normalized mean±SEM, **p<0.05 vs. PBS of Control, ^†p<0.05 vs. VEGF of Control, n=3 per group from 3 independent experiments); E) Quantification of migrated CECs on underside of Transwell insert in response to overnight treatment with 0.05% DMSO (Control) or 8CPT-AM (1 μM) in the presence of VEGF (50 ng/mL) or PBS (Normalized mean±SEM, **p<0.05 vs. PBS of Control, ^†p<0.05 vs. VEGF of Control, n=9 per group from 3 independent experiments); F) Representative confocal images of Vybrant-stained CECs on the underside of Transwell inserts in response to treatments described in Fig. 2E; G) Quantification of tube formation in CECs treated with 0.05% DMSO (Control) or 8CPT-AM (1 μM) in the presence of VEGF (50 ng/mL) or PBS for 12 hours (Normalized mean±SEM, **p<0.05 vs. PBS of Control, ^†p<0.05 vs. VEGF of Control, n=9 per group from 3 independent experiments); H) Representative fluorescent images of tube formation using Vybrant-stained CECs grown on top of Matrigel in response to treatments described in Fig. 2G

Fig. 3. Active Rap1 binding the IQ domain of IQGAP1 is necessary to reduce VEGF-induced Rac1 activation in CECs.

A) Representative western blot images that depict Rac1 activation by immunoprecipitation of Rac1GTP and blot of total Rac1 (row 1), total Rac1 (row 2), GFP-tagged IQGAP1 plasmid construct (i.e., either GFP-IQ-WT or GFP-IQ-ΔIQ) interactions with Rac1 by co-immunoprecipitation (row 3), GFP-tagged IQGAP1 pulled down by immunoprecipitation as loading control (row 4), Rap1 activation by immunoprecipitation (row 5), and total Rap1 (row 6) in lysates from CECs transfected with GFP-IQ-WT (columns 1–4) or GFP-IQ-ΔIQ (columns 5–8) and treated with 0.05% DMSO or 8CPT-AM (1 μM) in the presence VEGF (50 ng/mL) or PBS; B) Quantification of Rac1 activation by densitometry analysis (Normalized mean±SEM, ^†p<0.05 vs. Control of GFP-IQ-ΔIQ, *p<0.05 vs. VEGF of GFP-IQ-WT, ^‡p<0.05 vs. 8CPT-AM of GFP-IQ-WT, ^$p<0.05 vs. VEGF+8CPT-AM of GFP-IQ-WT, n=3 per group from 3 independent experiments); C) Representative confocal images of CECs labelled to identify GFP tagged IQGAP1 (green), Rap1GTP (red), and DAPI (blue) in response to 1 μL PBS and 0.05% DMSO (control), 50 ng/mL VEGF and 0.05% DMSO (VEGF), 1 μL PBS and 1 μM 8CPT-AM (8CPT-AM), or 50 ng/mL VEGF and 1 μM 8CPT-AM (VEGF+8CPT-AM); D) Quantification of Rap1GTP fluorescent density in response to treatments described in Fig. 3C (Normalized mean±SEM, **p<0.05 vs. Control of GFP-IQ-WT, ^††p<0.05 vs. Control of GFP-IQ-ΔIQ, n=3 per group from 3 independent experiments)

Fig. 4. Active Rap1 binding the IQ domain of IQGAP1 is sufficient to reduce VEGF-induced Rac1 activation in CECs.

A) Representative western blot images that depict Rac1 activation by immunoprecipitation of Rac1GTP and blot of total Rac1 (row 1), total Rac1 (row 2), Rac1GTP interactions with IQGAP1 by co-immunoprecipitation (row 3), total IQGAP1 (row 4), Rap1 activation to total Rap1 by co-immunoprecipitation (row 5), and total Rap1 (row 6) in lysates from CECs transfected with Myc-IQ-WT (columns 1–4) or Myc-IQ-3,4R (columns 5–8) and treated with 0.05% DMSO or 8CPT-AM (1 μM) in the presence VEGF (50 ng/mL) or PBS; B) Quantification of Rac1 activation by densitometry analysis (Normalized mean±SEM, ^#p<0.05 vs. Control of Myc-IQ-WT, *p<0.05 vs. VEGF of Myc-IQ-WT, ^‡p<0.05 vs. 8CPT-AM of Myc-IQ-WT, ^$p<0.05 vs. VEGF+8CPT-AM of Myc-IQ-WT, n=3 per group from 3 independent experiments)

Fig. 5. Activation of Rap1a is sufficient to prevent VEGF-induced Rac1 activation, migration, and tube formation in CECs.

A) Representative images of CECs transduced with adenovirus expressing GFP (Ad-GFP) or constitutively active Rap1a (Ad-GFP-63E) 48 hours post-infection; B) Representative western blot images that depict expression of GFP (columns 1 and 2) or constitutively active Rap1a (columns 3 and 4) following 48 hours of transduction and treatment with equal volumes of PBS or VEGF (50 ng/mL) for 30 minutes; C) Representative western blot images that depict Rac1GTP interactions with IQGAP1 by co-immunoprecipitation (row 1), total IQGAP1 (row 2), Rac1 activation by immunoprecipitation of Rac1GTP and blot of total Rac1 (row 3), and total Rac1 (row 4) in lysates from CECs transduced with Ad-GFP (columns 1 and 2) or Ad-GFP-63E (columns 3 and 4) and treated with PBS (columns 1 and 3) or VEGF (50 ng/mL, columns 2 and 4) for 30 minutes; D) Quantification of Rac1 activation by densitometry analysis (Normalized mean±SEM, *p<0.05 vs. PBS of Ad-GFP, ^††p<0.01 vs. VEGF of Ad-GFP, n=3 per group from 3 independent experiments); E) Quantification of transduced CECs migrating in response to overnight treatment with VEGF (50 ng/mL) or PBS (Normalized mean±SEM, *p<0.01 vs. PBS of Ad-GFP, ^††p<0.01 vs. VEGF of Ad-GFP, n=9 per group from 3 independent experiments); F) Representative confocal images of transduced CECs on the underside of Transwell insert in response to treatments described in Fig. 5E; G) Quantification of tube formation in transduced CECs treated with PBS or VEGF (50 ng/mL) for 12 hours (Normalized mean±SEM, *p<0.05 vs. PBS of Ad-GFP, ^††p<0.01 vs. VEGF of Ad-GFP, n=9 per group from 3 independent experiments); H) Representative confocal images of tube formation using transduced CECs in response to conditions described in Fig. 5G

Fig. 6. Characterization of tamoxifen-inducible endothelial IQGAP1 knockout mouse model.

A) Diagram of floxed (top) and recombined (bottom) Iqgap1 alleles that depict the locations of the designed primers for genotyping; B) PCR product demonstrating recombination of the floxed Iqgap1 allele in tamoxifen-injected IQ^iΔEC (bottom row, 2^nd lane) and not in tamoxifen-injected IQ^fl mice (bottom row, 1^st lane) using isolated DNA from ear punches; C) Representative images of live fluorescence imaging of the retina using the Micron IV that depict expression of YFP in endothelial cells in in tamoxifen-injected IQ^iΔEC (right, yellow arrow) and not in tamoxifen-injected IQ^fl mice (left, yellow arrow); D) Representative confocal images of RPE/choroid/scleral tissues that demonstrate Cre-mediated recombination in tamoxifen-injected IQ^iΔEC (right image) that were heterozygous for Cdh5-CreERT2 allele compared to no recombination in IQ^fl that lack Cdh5-CreERT2 (left image).Tamoxifen-injected IQ^iΔEC show loss of IQGAP1 (white) co-localization with lectin-stained vessels (red) and expression of YFP in the choroid (right image), whereas tamoxifen-injected IQ^fl demonstrated IQGAP1 (white) co-localization with lectin-stained vessels (red) without YFP expression (left image)

Fig. 7. Endothelial IQGAP1 is necessary for Rac1GTP-mediated CNV in the murine laser-induced CNV model.

A) Representative images of lectin-stained RPE/choroid flat mounts from IQ^fl showing no YFP and littermate IQ^iΔEC showing YFP; B) Quantification of laser-induced CNV volume (Mean±SEM, *p<0.05 vs. IQ^fl, n=36 CNV lesions from 9 different mice for IQ^fl and n=49 CNV lesions from 13 different mice; C) Representative confocal images of posterior eye cup sections that were co-labeled with antibodies against Rac1GTP (green), lectin (red), and TOPRO-3 (grey) in IQ^fl (middle column) and IQ^iΔEC (right column) at either 20X (top row) or 100X (bottom row) objective, staining with secondary antibody along with lectin and TOPRO-3 (left) was also performed in the absence of primary Rac1GTP antibody to determine non-specific fluorescence from secondary antibody in CNV lesion (white arrow); D) Quantification of Rac1GTP fluorescent density in lectin-labeled CNV lesion (Normalized mean±SEM, *p<0.05 vs. IQ^fl, n=6 sections from 3 different mice per group); (GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer)

Fig. 8. Activation of Rap1 reduces CNV in the murine laser-induced CNV model by interacting with endothelial IQGAP1.

A) Representative confocal images of lectin-stained RPE/choroid flat mounts from IQ^fl and littermate IQ^iΔEC treated with either intravitreal PBS (first column) or intravitreal 8CPT (second column); B) Quantification of laser-induced CNV volume (Mean±SEM, *p<0.05 vs. intravitreal PBS treated IQ^fl, n=44 CNV lesions from 11 different IQ^fl treated with intravitreal PBS, n=48 CNV lesions from 12 different IQ^fl treated with intravitreal 8CPT, n=32 CNV lesions from 8 different IQ^iΔEC treated with intravitreal PBS, n=56 CNV lesions from 14 different IQ^iΔEC treated with intravitreal 8CPT)

Fig. 9. Diagram of proposed mechanism in which active Rap1 interferes with Rac1 activation.

8CPT-induced active Rap1 binds to the IQ domain of IQGAP1 in choroidal endothelial cells. This interaction between active Rap1 and IQGAP1 interferes with active Rac1 binding to IQGAP1, which prevents sustained Rac1 activation. This results in reduced CEC migration and reduced development of CNV (the diagram was created with BioRender)