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Haibo Wang, Xing Yu, M Elizabeth Hartnett; IQGAP1 regulates the interaction of VEGFR2 and CCR3 in Rac1 mediated choroidal endothelial cell migration . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2047.
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© ARVO (1962-2015); The Authors (2016-present)
We previously found crosstalk between the C-C chemokine receptor type 3 (CCR3) and VEGFR2 in Rac1-mediated choroidal endothelial cell (CEC) migration, a necessary step in the development of choroidal neovascularization in neovascular AMD. IQGAP1, a scaffold protein with a Rac1 binding domain, can regulate receptor interactions and signaling involved in cell migration and adhesion. We tested the hypothesis that IQGAP1 mediates the interaction of ligand-activated VEGFR2 and CCR3 to cause Rac1 activation and CEC migration in cultured human CECs. <br />
Co-immunoprecipitation (Co-IP) of IQGAP1 and CCR3 or IQGAP1 and VEGFR2 was performed in CECs stimulated with VEGF (20 ng/mL), CCR3 ligand (CCL11, 10 ng/mL), both (VEGF+CCL11), or PBS control. Co-IP of VEGFR2 and CCR3, western blots of VEGFR2, CCR3 and IQGAP1, and Pull down assays of Rac1 activation were performed in CECs transfected with small interference RNA (siRNA) to human IQGAP1 gene and treated with CCL11, VEGF, CCL11+VEGF, or PBS control. CECs transfected with IQGAP1 siRNA or control siRNA were stimulated with CCL11, VEGF, CCL11+VEGF, or PBS control, and CEC migration was measured. Silencer selective negative siRNA and PBS treatment were used as controls. Experiments included 3 samples/condition, and each experiment was performed 3 times. Statistics were performed using ANOVA with post hoc protected Bonferroni Multiple Comparison Test and a minimum P value of <0.05 was considered statistically significant.<br />
In CECs, CCL11, VEGF or CCL11+VEGF induced interactions between IQGAP1 and CCR3 or IQGAP1 and VEGFR2. Compared to CCL11 or VEGF treatment alone, CCL11+VEGF caused a 25% greater interaction between VEGFR2 and CCR3 (p<0.05), a 3 fold greater Rac1 activation (p<0.05), and significantly greater CEC migration (p<0.05). Knockdown of IQGAP1 in CECs by siRNA transfection significantly inhibited VEGF+CCL11 induced co-immunoprecipitation of CCR3 and VEGFR2, VEGFR2 induced Rac1 activation, and CEC migration (p<0.01). <br />
IQGAP1 mediates the interaction between activated CCR3 and VEGFR2 and VEGF-induced CEC migration mediated through Rac1. Future studies are indicated to test the role of IQGAP1 in choroidal neovascularization in vivo and may provide insight into mechanisms involved in neovascular AMD.
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