Abstract
Purpose :
Endomucin (EMCN) is a type I integral membrane glycoprotein selectively expressed by venous and capillary endothelium. Our previous findings showed that EMCN knockdown significantly inhibits VEGF165-induced VEGFR2 internalization and downstream activities. The goal of this study is to further define the specificity of EMCN for VEGF/VEGFR2 system by determining the role of EMCN in VEGF121-induced VEGFR2 activation. We also examined the potential role of EMCN in fibroblast growth factor (FGF) signaling and angiogenesis-related activities in endothelial cells.
Methods :
EMCN was knocked down in human retinal endothelial cells (HRECs) using siEMCN, with non-targeting siRNA as a control. Whole cell lysates of HRECs with or without EMCN knockdown, followed by VEGF165 or VEGF121 stimulation at 5, 10, 30 and 60 min, were harvested. Protein levels of total VEGFR2, phospho-VEGFR2 (Y1175), total Akt, phospho-Akt, total Erk, phospho-Erk, total Src and phospho-Src were examined by western blot. A wound-healing assay was used to examine endothelial cells migration.
Results :
VEGF165 and VEGF121 stimulation significantly increased HRECs wound closure compared to control (1 ±0.02 vs. 1.15 ±0.02, p=0.004; 1 ±0.02 vs. 1.18±0.03, p=0.0001. N=3 for both). SiEMCN treatment led to a significant suppression of EMCN protein levels compared to non-targeting siRNA group by 95% (P<0.05). EMCN knockdown prevented HRECs migration induced by VEGF165 (1 ±0.03 vs. 1.04 ±0.03, p=0.9, n=3) as well as VEGF121 compared to control (1 ±0.03 vs. 1.07 ±0.02, p=0.5, n=3). The levels of phospho-Src significantly increased following VEGF121 stimulation for 10 min (n=3, p<0.05); EMCN knockdown inhibited phospho-Src expression compared control (n=3, p<0.05). No significant differences were detected between EMCN knockdown and control for the expression of phospho-VEGFR2, phospho-Akt and phospho-Erk. FGF stimulation significantly increased HRECs wound-closure (n=6, p<0.0001), but EMCN knockdown did not significantly affect FGF-induced HRECs migration compared to control (n=6, p<0.001).
Conclusions :
EMCN is essential for VEGF165- and VEGF121-induced endothelial migration and VEGR2 internalization. However, EMCN does not play a significant role in FGF-induced endothelial cells migration. Our data indicate a specific role for EMCN in the VEGF/VEGFR2 system.
This is a 2021 ARVO Annual Meeting abstract.