Abstract
BackgroundEndothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis.ResultsIn human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls.ConclusionsThese results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.
Highlights
Endothelial intercellular junctions play an essential role in many critical processes, including survival, angiogenesis and inflammation [1,2,3]
Localization of intercellular adhesion molecule (ICAM)-2 and NCad on human endothelial cells (EC) in sub-confluent vs confluent monolayers The organization of adherens junctions (AJ) can be different at different stages of confluence and maturity; we set out to investigate the localization of ICAM-2 and the two cadherins on EC by performing co-staining in human umbilical vein endothelial cells (HUVEC) monolayers at different stages of confluency
These data indicate that in sub-confluent HUVEC monolayers both VECad and NCad can be found at the junctions, and suggest that the localization and expression of Ncadherin in endothelial cells is partly due to the maturation stage of adherens junctions
Summary
Endothelial intercellular junctions play an essential role in many critical processes, including survival, angiogenesis and inflammation [1,2,3]. Complex signaling networks regulated by cell-cell junctions control contact inhibition, cell growth, survival and permeability [2,4]. These pathways are modulated by cross-talk between different junctional adhesion molecules and with other surface receptors. VECad expression is restricted mainly to endothelial cells (EC), whilst NCad is expressed in several cell types including neurons, muscle cells and fibroblasts [6,7,8,9] Both cadherins are involved in vascular development and angiogenesis: endothelial deletion of either VECad or NCad leads to embryonic lethality due to vascular defects [10,11]. The intercellular adhesion molecule (ICAM)-2, localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis
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