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Zhengping Hu, Michelle LeBlanc, kahira Saez-Torres, Magali Saint-Geniez, Yin Shan Eric Ng, Patricia A. D'Amore; Endomucin regulates Vascular Endothelial Growth Factor (VEGF) induced endothelium functions through its extracellular domain. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5376.
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Endomucin (EMCN) is an endothelial cell-specific type I integral membrane glycoprotein. Our previous data suggest that EMCN knockdown significantly impairs retinal vessel development in vivo, and prevents VEGF-induced cell proliferation, migration and tube formation in vitro. It interacts with VEGFR2 in a VEGF dependent fashion, and modulates VEGFR2 downstream signaling. The goal of this study is to identify the structural aspects of EMCN that are necessary for VEGF-induced endothelial cell functions.
Mouse EMCN mutants with truncated extracellular domains were generated (Δ21-81, Δ21-121 and Δ21-161) and expressed using pAdEasy in human retinal endothelial cells (HRECs) in which endogenous EMCN had been knocked down using siRNA against human EMCN. Non-targeting siRNA was the control for knockdown. Knockdown of endogenous EMCN in HRECs expressing truncated mutants was confirmed by qPCR. Cell surface expression was determined by labeling with NHSS-SS biotin and visualization by western blot. The ability of the truncation mutants to rescue VEGF-induced activity was accessed in cell migration and tube formation assays.
Endogenous EMCN was significantly knocked down at the mRNA level in HRECs expressing mouse mutants (p<0.001). The peak expression of mouse truncation mutants following adenovirus transduction in the HRECs was at 24-48 h. Cell surface expression of EMCN mutants was confirmed by western blot. Expression of mouse EMCN FL, Δ21-81 and Δ21-121 in HRECs in which endogenous EMCN was knocked down rescued cell migration following VEGF stimulation, compared to mCherry control (fold change: 1.7±0.04, 1.43±0.08, 1.46±0.96, respectively. N=8). EMCN Δ21-81 and Δ21-121 rescued VEGF-stimulated tube formation (total tube length and total segment length) in HRECs (p<0.05, N=3). In contrast, expression of EMCN Δ21-161 was unable to rescue VEGF-induced HREC migration (fold change: 1.13±0.087) or tube formation (fold change: 0.88±0.06), compared to mCherry.
Our data indicate an essential role for amino acids 121-161 of EMCN’s extracellular domain in mediating VEGF-induced EC migration and tube formation. These studies reveal the EMCN extracellular domain as a potential target for VEGF-induced angiogenesis. Its endothelial cell specificity and the fact that it targets VEGFR2 may represent advantages over currently available therapies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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