Person:

Yang, Jinling

Endomucin is a membrane-bound glycoprotein expressed luminally by endothelial cells that line postcapillary venules, a primary site of leukocyte recruitment during inflammation. Here we show that endomucin abrogation on quiescent endothelial cells enables neutrophils to adhere firmly, via LFA-1-mediated binding to ICAM-1 constitutively expressed by endothelial cells. Moreover, TNF-α stimulation downregulates cell surface expression of endomucin concurrent with increased expression of adhesion molecules. Adenovirus-mediated expression of endomucin under inflammatory conditions prevents neutrophil adhesion in vitro and reduces the infiltration of CD45+ and NIMP-R14+ cells in vivo. These results indicate that endomucin prevents leukocyte contact with adhesion molecules in non-inflamed tissues and that downregulation of endomucin is critical to facilitate adhesion of leukocytes into inflamed tissues.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and hemostasis1. Hemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here, using an engineered organotypic model of perfused microvessels and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain – the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling.

Angiogenesis is central to both normal and pathologic processes. Endothelial cells (ECs) express O-glycoproteins that are believed to play important roles in vascular development and stability. Endomucin-1 (EMCN) is a type I O-glycosylated, sialic-rich glycoprotein, specifically expressed by venous and capillary endothelium. Evidence has pointed to a potential role for EMCN in angiogenesis but it had not been directly investigated. In this study, we examined the role of EMCN in angiogenesis by modulating EMCN levels both in vivo and in vitro. Reduction of EMCN in vivo led to the impairment of angiogenesis during normal retinal development in vivo. To determine the cellular basis of this inhibition, gain- and loss-of-function studies were performed in human retinal EC (HREC) in vitro by EMCN over-expression using adenovirus or EMCN gene knockdown by siRNA. We show that EMCN knockdown reduced migration, inhibited cell growth without compromising cell survival, and suppressed tube morphogenesis of ECs, whereas over-expression of EMCN led to increased migration, proliferation and tube formation. Furthermore, knockdown of EMCN suppressed VEGF-induced signaling as measured by decreased phospho-VEGFR2, phospho-ERK1/2 and phospho-p38-MAPK levels. These results suggest a novel role for EMCN as a potent regulator of angiogenesis and point to its potential as a new therapeutic target for angiogenesis-related diseases.