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Michelle LeBlanc, Kahira Saez-Torres, Magali Saint-Geniez, Yin Shan Eric Ng, Patricia A D'Amore; Endomucin (EMCN) Regulates Angiogenesis by Controlling VEGFR2 Endocytosis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5481. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glycoproteins regulate angiogenesis by promoting endothelial cell (EC) survival, proliferation and migration. Endomucin (EMCN) is an integral membrane glycoprotein expressed on the surface of ECs, and was identified by our lab as a novel regulator of angiogenesis. EMCN knockdown in vitro prevents vascular endothelial growth factor (VEGF)-induced proliferation and migration, whereas overexpression enhances these effects. The purpose of this work is to define the mechanism through which EMCN modulates VEGF-induced angiogenesis.
EMCN knockdown was achieved in human retinal microvascular ECs (HRECs) using siRNA against human EMCN (siEMCN, GE Healthcare). Endocytosis was evaluated using an immunocytochemistry (ICC) internalization assay and quantified by measuring intracellular fluorescence intensity. HRECs were incubated with anti-VEGF receptor 2 (VEGFR2, R&D) or anti-EMCN (Abcam) at 4oC and stimulated with VEGF (10 ng/ml) for 30 min at 37oC. Cell surface antibodies were removed with a low pH buffer. Internalized antibodies were detected using fluorescent secondary antibodies. For cell surface labeling, ECs were incubated with NHS-SS-Biotin (Life Technology), isolated proteins were resolved using western blot, and probed with antibodies against VEGFR2 (Cell Signaling), EMCN and CD31 (Abcam). ICC was conducted using antibodies against EMCN and CD31.
Western blot verified EMCN knockdown using siRNA (n=3, p<0.01). ICC internalization results showed an increase in VEGF-induced VEGFR2 internalization in control HRECs (n=4, p<0.05). EMCN was not co-internalized under these conditions. In the absence of EMCN, VEGF-induced VEGFR2 internalization was reduced (n=6, p<0.001). siECMN had no effect on basal VEGFR2 endocytosis (n=6). Biotin-labeled surface VEGFR2 was quantified using western blot, and decreased following VEGF stimulation in control but not in siEMCN transfected HRECs (n=5, p<0.01). ICC on non-permeabilized VEGF-stimulated ECs confirmed that in the absence of EMCN, VEGFR2 was retained on the cell surface.
Our results demonstrate that EMCN modulates VEGF-induced, but not basal, VEGFR2 endocytosis. These findings are significant in that VEGFR2 internalization is an essential step in VEGF-induced signaling. Thus, loss of EMCN can dampen VEGFR2 signaling and downstream activity. These findings suggest that inhibition of EMCN may be an EC-specific therapy for targeting VEGFR2 activity.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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