Endomucin regulates both VEGFR2 and VEGFR1 internalization by human retinal microvascular endothelial cells
Kuo, Blanche   (Massachusetts General Hospital)
Mentor: D’Amore, Patricia A. (Harvard Medical School)
Introduction: Angiogenesis is involved in many pathologic conditions, such as tumor vascularization and diabetic retinopathy. Our laboratory has demonstrated a role for the glycocalyx, particularly endomucin (EMCN), in the regulation of angiogenesis. To date, we have shown that EMCN regulates the activity of VEGFR2. However, VEGFR1, which interacts with VEGF and with VEGFR2, also plays a central role angiogenesis, but less is known about its regulation. Here, we aim to examine the effect of EMCN on VEGFR1 and its role of angiogenesis. Hypothesis: Given that EMCN is required for VEGFR2 internalization and clathrin-mediated endocytosis, we hypothesize that EMCN is required for VEGFR1 internalization. Methods: VEGFR1 expression by human retinal microvascular endothelial cells (HRECs) was analyzed using western blot probed with using anti-VEGFR1 (Sigma). Immunocytochemistry was conducted using anti-VEGFR2 (R&D), rat anti-mEMCN (L6H10) and anti-Rab5 (Cell Signaling). Biotinylation was employed to examine cell surface proteins. VEGFR1 and EMCN were knocked down using siRNA directed against VEGFR1 (siVEGFR1), EMCN (siEMCN) or non-targeting control (siNT). HRECs were stimulated with VEGF-165 (10 ng/mL, R&D) or PlGF-2 (10 ng/mL and 250ng/mL, R&D). VEGFR1phosphorylation was assessed by western blot using anti-phospho-VEGFR1 (Millipore). ICC based internalization assay was used to assess VEGFR1 internalization. Results: Western blot analysis revealed that HRECs expressed both membrane and soluble isoforms of VEGFR1. Significant knockdown of the VEGFR1 soluble isoform was achieved using siVEGFR1 compared to siNT control at 48h (p<0.05). Immunocytochemistry showed higher levels of intracellular VEGFR1 compared to cell surface VEGFR1. VEGF stimulation induced both VEGFR1 and VEFGR2 internalization after 30 min. Both VEGF-165 and PlGF-2 induced VEGFR1 internalization after 30 min of stimulation. EMCN depletion in HRECs significantly reduced VEGF (p<.05) and PlGF (p<.01) induced VEGFR1 internalization. PlGF stimulation for 5 min activated VEGFR1 as seen by an increase in phosphorylation of VEGFR1 (Y1213) detected by western blot. Conclusions: These findings are similar to previous results showing that EMCN depletion preventing VEGFR2 internalization, suggesting that EMCN is essential in receptor internalization of both VEGFR1 and VEGFR2 with therapeutic potential.