The Effect of Endomucin on Cell Surface Receptor Signaling Is Specific for Vascular Endothelial Growth Factor Receptor 2.

Endomucin (EMCN) is a mucin-like glycoprotein selectively expressed by venous and capillary endothelium and, as a component of the endothelial glycocalyx, inhibits leukocyte adhesion. Previously our lab has determined that EMCN interacts with VEGFR2 and that its depletion inhibits VEGF165-induced VEGFR2 clathrin-mediated endocytosis and angiogenesis-related functions. We examined whether other tyrosine kinase receptors required the presence of EMCN or if EMCN regulates specifically VEGF165-induced VEGFR2 signaling. Thus, we investigated the role of EMCN in receptor internalization and signaling for VEGF121/VEGFR2, VEGF165/VEGFR1, and basic fibroblast growth factor (FGF-2)/FGF receptor 1 (FGFR1). Human retinal microvascular endothelial cell (HREC) migration was determined using a scratch wound-healing assay in which the extent of cell migration over a 16 hr period was quantified. EMCN mRNA depletion (95%, P<0.05) was achieved using siRNA. Equal molar concentration of VEGF165 (10 ng/ml) and VEGF121 (7.28 ng/ml), and FGF-2 at 10 ng/ml induced significant migration of HRECs compared to untreated controls (1.15 ± 0.02 vs. 1 ± 0.02, P=0.0027, 1.19 ± 0.03 vs. 1 ± 0.02, P<0.0001, 1.25 ± 0.04 vs. 1 ± 0.03, P<0.0001, respectively and n=12 for all groups). EMCN knockdown resulted in a significant decrease in cell migration stimulated by VEGF165 and VEGF121 compared to control (1.01 ± 0.02 vs. 1.15 ± 0.04, P=0.0061, 1.04 ± 0.04 vs. 1.19 ± 0.05, P=0.0021, n=12). EMCN knockdown did not decrease FGF-2-induced migration compared to control (1.218 ± 0.08 vs. 1.248 ± 0.06, P=1.0, n=12). To determine internalization of cell surface receptors HREC surface proteins were labeled using sulfo-NHS-SS biotin, isolated using neutral-avidin beads and analyzed by western blot. Obtained values represent remaining receptor at the cell surface. EMCN knockdown did not influence VEGF165-induced VEGFR1 internalization (0.625 ± 0.11 vs. 0.386 ± 0.08, P=0.11, n=6), or FGF-2-induced FGFR1 internalization (0.734 ± 0.075 vs. 0.769 ± 0.074, p=0.74 n=7). These data suggest that EMCN modulates VEGFR2 internalization and function induced by both VEGF165 and VEGF121 isoforms, but does not impact VEGF165-induced VEGFR1 or FGF-2-induced FGFR1 internalization and function. These data support that the effects of EMCN on angiogenesis occur specifically through modulation of VEGFR2 function.