Abstract
Purpose :
The endothelial glycocalyx, lining the apical surface of the endothelium, is involved in a host of vascular processes. The layer contains a network of membrane-bound proteoglycans and glycoproteins. One such glycoprotein is endomucin (EMCN), which our lab has revealed is a modulator of VEGFR2 function. Intravitreal injection of siEMCN into the eyes of P5 mice impairs vascular development. In vitro silencing of EMCN suppresses VEGF-induced proliferation and migration. Signaling pathways that drive cell migration converge on cytoskeletal remodeling. By coupling co-immunoprecipitation with liquid chromatography/mass spectrometry, we identified interactions between EMCN and proteins associated with actin cytoskeleton organization. The aim of the study is to investigate the influence of EMCN on cytoskeleton dynamics in angiogenesis.
Methods :
The effects of EMCN were evaluated ex vivo using a choroidal explant sprouting assay and in vitro by phalloidin staining in human retinal endothelial cells (HRECs). Following AAV-mediated Cre deletion in choroidal punches isolated from EMCN-floxed mice, explants were maintained for six days. Angiogenesis was quantified by measuring the vascular sprouting area. To knockdown EMCN, HRECs were transfected with siRNA directed against human EMCN or non-targeting siRNA. Post-transfection, serum starved HRECs were stimulated with VEGF. To overexpress EMCN, cells were infected with adenovirus expressing myc-tagged EMCN. Actin was stained with phalloidin conjugated to Alexa Fluor 594. Analyses of sprouting area and mean fluorescence intensity were performed in ImageJ.
Results :
The sprouting area was significantly reduced in choroidal explants with EMCN knockdown exposed to AAV-Cre (2.6±0.4 mm2) compared to tissues treated with AAV-GFP control (1.1±0.2 mm2). Western blot verified EMCN knockdown in HRECs. In the absence of VEGF, there were significantly lower absolute levels of F-actin. A comparable reduction in actin filaments was observed in siEMCN transfected cells stimulated with VEGF. Notably, overexpression of EMCN induced cell surface projections that coincided with an increased concentration of F-actin.
Conclusions :
Loss of EMCN inhibited mouse choroidal sprouting, and EMCN expression correlated with formation of F-actin containing stress fibers. Our findings support further study of EMCN’s role in endothelial cell rearrangement and cell shape changes during angiogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.