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. 2020 Nov;23(4):685-698.
doi: 10.1007/s10456-020-09740-y. Epub 2020 Aug 11.

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation

Haibo Wang  1 , Aniket Ramshekar  1 , Eric Kunz  1 , David B Sacks  2 , M Elizabeth Hartnett  3
Affiliations

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation

Haibo Wang et al. Angiogenesis. 2020 Nov.

Abstract

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1+/+, Iqgap1-/- mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

Keywords: Age-related macular degeneration; IQGAP1; Rac1; Vascular endothelial growth factor; Vascular endothelial growth factor receptor 2; choroidal neovascularization.

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Conflict of interest statement

Conflicts of Interests

The authors declared there were no conflicts of interests to disclose.

Figures

Fig. 1.
Fig. 1.. Isoforms of IQGAP family proteins: IQGAP1, IQGAP2 and IQGAP3.
All the isoforms contain a GRD domain.
Fig. 2.
Fig. 2.. IQGAP1 is located at CNV lesions.
(a) Immunostaining of IQGAP1 in retinal sections of human donor eyes with AMD (Red: IQGAP1; Gray: Topro 3 to stain nuclei); (b) immunostaining of IQGAP1 and isolectin in retinal cryosections of wild type mice treated with laser (Green: IQGAP1; Red: isolectin; Gray: Topro3 to stain nuclei; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; the white arrows point to type 2 CNV).
Fig. 3.
Fig. 3.. Iqgap1−/− mice show reduced CNV in a laser-induced CNV model.
Western blots of (a) IQGAP1, (b-c) IQGAP2, and (d-e) IQGAP3 in RPE/choroids of Iqgap1+/+ and Iqgap1−/− mice 7 days post laser treatment (b and d, representative gel images, and c and e, quantification of densitometry); (f) Representative images of RPE/choroid flat mounts and (g) quantification of CNV lesion (*p<0.05 vs. Iqgap1+/+ mice, n= 52 spots from 14 mice) in Iqgap1+/+ and Iqgap1−/− mice 7 days post laser treatment.
Fig. 4.
Fig. 4.. Iqgap1−/− mice show increased VEGF and decreased VEGFR2 and Rac1 activation in a laser-induced CNV model.
(a and b) western blots of VEGF and β-actin, (c and d) co-immunoprecipitation of VEGFR2 (p-VEGFR2) and western blots of p-Tyr, total VEGFR2 and β-actin in RPE/choroids, and (e and f) immunostaining of GTP-Rac1, isolectin and Topro 3 in retinal cryosections (Green: GTP-Rac1; Red: isolectin; Gray: Topro 3; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer) of Iqgap1+/+ and Iqgap1−/− mice 7 days post laser treatment (a, c and e, representative gel images and b, d and f, quantification of densitometry and fluorescence density of GTP-Rac1 at isolectin stained CNV lesions; *p<0.05, ***p<0.001 vs. Iqgap1+/+ mice; n= 6–8; white arrows point to type 2 CNV).
Fig. 5.
Fig. 5.. IQGAP1 regulates VEGF-mediated VEGFR2 activation and Rac1 activation.
(a–b) co-immunoprecipitation of VEGFR2 and IQGAP1 and phosphorylated VEGFR2 (p-VEGFR2) and western blots of total VEGFR2, IQGAP1, and β-actin in CECs transfected with IQGAP1 siRNA or control siRNA and treated with VEGF for 15 mins; (c–d) co-immunoprecipitation of GTP-Rac1 and IQGAP1, and GTP-Rac1 determined by immunoprecipitation with anti-GTP-Rac1 and western blot of total Rac1 in CECs transfected with IQGAP1 siRNA or control siRNA and treated with VEGF for 30 mins; (e) cell migration toward VEGF of CECs transfected with IQGAP1 siRNA or control siRNA and (f) tube formation induced by VEGF of CECs transduced with adenovirus-delivered IQGAP1shRNA or controlshRNA (**p<0.01, ***p<0.001vs. PBS of controlsiRNA, ††p< 0.01 vs. PBS of IQGAP1siRNA, ‡p<0.05, ‡‡‡p<0.001 vs. VEGF of ControlsiRNA; ##p<0.01 vs. PBS of ControlshRNA and $ $p<0.01 vs. VEGF of ControlshRNA; n=3–6).
Fig. 6.
Fig. 6.. Direct binding of Rac1GTP to GRD of IQGAP sustains Rac1 activation.
Co-immunoprecipitation of GTP-Rac1 and IQGAP1, and GTP-Rac1 determined by immunoprecipitation with anti-GTP-Rac1 and western blot of total Rac1 in CECs transfected with plasmid DNAs expressing full length IQGAP1 (GFP-IQ-WT) or mutant GRD (GFP-IQ-MK24) and treated with VEGF for for 15, 30, 120 mins or PBS control (a, representative gel images, and b, quantification of densitometry; **p<0.01, ***p<0.001 vs. PBS of GFP-IQ-WT, †††p<0.001 vs. PBS of GFP-IQ-MK24; n=3).
Fig. 7.
Fig. 7.. IQGAP1 regulates Rac1 activation by mediating Src activation.
(a and b) Western blots of phosphorylated Src (p-Src), total Src and β-actin in RPE/choroids of Iqgap1+/+ and Iqgap1−/− mice 7 days post laser treatment (*p<0.05 vs. Iqgap1+/+ mice; n=6–8; a, representative gel images and b, quantification of densitometry); (c-d) western blots of p-Src, total Src, IQGAP1 and β-actin and co-immunoprecipitation of VEGFR2 and Src, and western blots of total VEGFR2, p-Src total Src and β-actin in CECs transfected with IQGAP1 siRNA or control siRNA and treated with VEGF for 15 or 30 mins; and (e-f) activation of Rac1 determined by immunoprecipitation of anti-GTP-Rac1 and western blot of total Rac1, and western blots of total Rac1, p-Src, total Src and β-actin in CECs pretreated with Src inhibitor, Saracatinib (10 nM), for 30 mins followed by VEGF treatment for 30 or 60 mins (††p<0.01 vs. PBS of ControlsiRNA, ‡‡‡p<0.001 vs. PBS of IQGAP1siRNA, $ $p<0.01, $ $ $p<0.001 vs. PBS of Control, n=3; c and e, representative gel images, and d and f, quantification of densitometry).
Fig. 8.
Fig. 8.. Inhibition of Src or Rac1 inhibits VEGF-mediated CEC migration and tube formation.
a. CEC migration and b. tube formation were performed in CECs incubated with Src inhibitor, Saracatinib (10 nM), or/and Rac1 inhibitor (50 μM) in the presence or absence of VEGF (25 ng/ml) overnight (*p<0.05, **p<0.01 vs. PBS of Control; p=0.06, †p<0.05, ††p<0.01, and †††p<0.001 vs. VEGF of Control; #p<0.05 vs. PBS of Rac1 inhibitor and $ $p<0.01 vs. VEGF of Rac1 inhibitor; n=6–9 from two individual experiments).
Fig. 9.
Fig. 9.. Diagram of signaling pathway of IQGAP1 in regulation of VEGFR2-Src-Rac1 activation in choroidal angiogenesis.
In choroidal endothelial cell, upon binding to VEGF, VEGFR2 interacts with IQGAP1, leading to VEGFR2 activation. Activated VEGFR2 recruits Src and results in Src phosphorylation. Once Src is activated by VEGFR2 and IQGAP1, it activates Rac1 potentially involving phosphorylation of vav guaninenucleotide exchange factor 2 (Vav2), then leads to Src activation (from Rac1GDP to Rac1GTP). Once Rac1 is activated, Rac1GTP binds to IQGAP1 via GRD domain. This interaction between IQGAP1 and Rac1 maintains persistent Rac1 activation, which leads to CEC migration and CNV formation (the diagram was produced with BioRender).
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