525.5 - The Effect of Endomucin on Cell Surface Receptor Signaling Is Specific for Vascular Endothelial Growth Factor Receptor 2
Saturday, April 2, 2022
12:20 PM – 12:23 PM
Room: 118 BC - Pennsylvania Convention Center
Introduction: Session Description: As a Society, we cannot escape the identity crisis we have confronted in the past - what is pathology and how do pathologists fit into the basic framework of biomedical science? This is an ongoing challenge that requires our members to educate others regarding the nature of the discipline of experimental pathology and how our research describes and investigates the pathology, pathogenesis, and pathophysiology of specific diseases at the molecular, cellular, organ, and organismal level. Overcoming this identity crisis requires effort on the part of each ASIP member and our success will be evident as we continue to attract bright and enthusiastic young investigators into the diverse field of experimental pathobiology.
The American Society for Investigative Pathology presents I Am An ASIP Member and This Is My Science a dynamic and inspiring session featuring ASIP Scientists on the Cutting Edge of Discovery briefly, present their research, accomplishments, career journeys, and service to ASIP. This session highlights the diversity among our membership, and provides trainees, young scientists, pathologists, and the members of the larger scientific community the opportunity to become inspired by Trailblazers in the field of investigative pathology.
Issahy Cano (Schepens Eye Research Institute), Zhengping Hu (Schepens Eye Research Institute, Harvard Medical School), Magali Saint-Geniez (Schepens Eye Research Institute, Harvard Medical School), Yin Shan Ng (Schepens Eye Research Institute, Harvard Medical School), Patricia DAmore (Schepens Eye Research Institute, Harvard Medical School, Harvard Medical School)
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%, Plt;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, Plt;0.0001, 1.25 ± 0.04 vs. 1 ± 0.03, Plt;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.
Funding was provided by the 5R01EY026539-05 grant.
