Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

Novel engineered, membrane-tethered VEGF-A variants promote formation of filopodia, proliferation, survival, and cord or tube formation by endothelial cells via persistent VEGFR2/ERK signaling and activation of CDC42/ROCK pathways.

Therapeutic angiogenesis would be clinically valuable in situations such as peripheral vascular disease in diabetic patients and tissue reperfusion following ischemia or injury, but approaches using traditional isoforms of vascular endothelial growth factor-A (VEGF) have had little success. The isoform VEGF165 is both soluble and matrix-associated, but can cause pathologic vascular changes. Freely diffusible VEGF121 is not associated with pathologic angiogenesis, but its failure to remain in the vicinity of the targeted area presents therapeutic challenges. In this study, we evaluate the cellular effects of engineered VEGF variants that tether extracellular VEGF121 to the cell membrane with the goal of activating VEGF receptor 2 (VEGFR2) in a sustained, autologous fashion in endothelial cells. When expressed by primary human retinal endothelial cells (hRECs), the engineered, membrane-tethered variants eVEGF-38 and eVEGF-53 provide a lasting VEGF signal that induces cell proliferation and survival, increases endothelial permeability, promotes the formation of a cord/tube network, and stimulates the formation of elongated filopodia on the endothelial cells. The engineered VEGF variants activate VEGFR2, MAPK/ERK, and the Rho GTPase mediators CDC42 and ROCK, activities that are required for the formation of the elongated filopodia. The sustained, pro-angiogenic activities induced by eVEGF-38 and eVEGF-53 support the potential of engineered VEGF variants-overexpressing endothelial cells as a novel combination of gene and cell-based therapeutic strategy for stimulating endothelial cell-autologous therapeutic angiogenesis.

ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation in vitro and angiogenesis in vivo. In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function.

Background: Endomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. Methods: Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and α-smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. Results: EMCN -/- mice were viable with no gross anatomical defects in kidneys. The EMCN -/- mice exhibited increased infiltration of CD45 + cells, with an increased proportion of Ly6G high Ly6C high myeloid cells and higher VCAM-1 expression. EMCN -/- mice displayed albuminuria with increased albumin in the Bowman's space compared to the EMCN +/+ littermates. Glomeruli in EMCN -/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. Conclusion: Our study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

Update on the Role of the Endothelial Glycocalyx in Angiogenesis and Vascular Inflammation.

The endothelial glycocalyx is a negatively charged, carbohydrate-rich structure that arises from the luminal surface of the vascular endothelium and is comprised of proteoglycans, glycoproteins, and glycolipids. The glycocalyx, which sits at the interface between the endothelium and the blood, is involved in a wide array of physiological and pathophysiological processes, including as a mechanotransducer and as a regulator of inflammation. Most recently, components of the glycocalyx have been shown to play a key role in controlling angiogenesis. In this review, we briefly summarize the structure and function of the endothelial glycocalyx. We focus on its role and functions in vascular inflammation and angiogenesis and discuss the important unanswered questions in this field.

Galectin-3 Enhances Vascular Endothelial Growth Factor-A Receptor 2 Activity in the Presence of Vascular Endothelial Growth Factor.

Galectin-3 (Gal3) is a carbohydrate-binding protein reported to promote angiogenesis by influencing vascular endothelial growth factor-A receptor 2 (VEGFR2) signal transduction. Here we evaluated whether the ability of Gal3 to function as an angiogenic factor involved vascular endothelial growth factor (VEGF). To address this possibility we used human retinal microvascular endothelial cells (HRECs) to determine whether exogenous Gal3 requires VEGF to activate VEGFR2 signaling and if Gal3 is required for VEGF to activate VEGFR2. VEGFR2 phosphorylation and HREC migration assays, following either VEGF neutralization with ranibizumab or Gal3 silencing, revealed that VEGF endogenously produced by the HRECs was essential for the effect of exogenous Gal3 on VEGFR2 activation and cell migration, and that VEGF-induced VEGFR2 activation was not dependent on Gal3 in HRECs. Gal3 depletion led to no reduction in VEGF-induced cell function. Since Gal3 has been suggested to be a potential therapeutic target for VEGFR2-mediated angiogenesis, it is crucial to define the possible Gal3-mediated VEGFR2 signal transduction mechanism to aid the development of efficacious therapeutic strategies.

Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.