Abstract. In this paper we report that the assembly of interendothelial junctions containing the cell typespecific vascular endothelial cadherin (VE-cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells.Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, a,-catenin, and fl-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE-cadherin increased in closely packed monolayers.Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE-cadherin, ot-catenin, ~-catenin, and plakoglobin. All these proteins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junctional VE-cadherin, ot-catenin, and/3-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin coimmunoprecipitated with VE-cadherin decreased in migrating EC.These data suggest that VE-cadherin, ot-catenin, and /3-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascent cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, a-catenin, and/3-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VEcadherin/a-catenin//J-catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.

Human vascular endothelial cadherin (VE-cadherin, 7B4/cadherin-5) is an endothelial-specific cadherin localized at the intercellular junctions. To directly investigate the functional role of this molecule we cloned the full-length cDNA from human endothelial cells and transfected its coding region into Chinese hamster ovary cells. The product of the transfected cDNA had the same molecular weight as the natural VE-cadherin in human endothelial cells, and reacted with several VE-cadherin mouse monoclonal antibodies. Furthermore, it selectively concentrated at intercellular junctions, where it codistributed with alpha-catenin. VE-cadherin conferred adhesive properties to transfected cells. It mediated homophilic, calcium-dependent aggregation and cell-to-cell adhesion. In addition, it decreased intercellular permeability to high-molecular weight molecules and reduced cell migration rate across a wounded area. Thus, VE-cadherin may exert a relevant role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions.

Vascular endothelial cadherin (VE-cadherin, cadherin-5, or 7B4) is an endothelial specific cadherin that regulates cell to cell junction organization in this cell type. Cadherin linkage to intracellular catenins was found to be required for their adhesive properties and for localization at cell to cell junctions. We constructed a mutant form of VE-cadherin lacking the last 82 amino acids of the cytoplasmic domain. Surprisingly, despite any detectable association of this truncated VE-cadherin to catenin-cytoskeletal complex, the molecule was able to cluster at cell-cell contacts in a manner similar to wild type VE-cadherin. Truncated VE-cadherin was also able to promote calcium-dependent cell to cell aggregation and to partially inhibit cell detachment and migration from a confluent monolayer. In contrast, intercellular junction permeability to high molecular weight molecules was severely impaired by truncation of VEcadherin cytoplasmic domain. These results suggest that the VE-cadherin extracellular domain is enough for early steps of cell adhesion and recognition. However, interaction of VE-cadherin with the cytoskeleton is necessary to provide strength and cohesion to the junction. The data also suggest that cadherin functional regulation might not be identical among the members of the family.Cadherins are a family of integral membrane glycoproteins responsible for homotypic calcium-dependent cell-cell adhesion (1-3). All members of the family present strong homology at the amino acidic level (4 -9), but the degree of conservation differs with the different domains of the molecules. The cytoplasmic domain presents the highest conservation level and is involved in the binding to cytoplasmic proteins called catenins, that contribute to the anchorage of cadherins to the cytoskeletal network (10 -15).Previous studies have shown that deletions or substitutions of the cytoplasmic domain of cadherins results in loss of association to catenins and in inhibition of cell aggregation (12, 16). Furthermore, recent studies indicate that tyrosine phosphorylation of cadherins and/or catenins decreases the amount of actin-bound complexes, and this was related to impairment of cadherin-mediated cell to cell aggregation and increased cell mobility (17)(18)(19). Most of these observations were made taking E-cadherin (or uvomorulin) as a model. However, considering other cadherins, the requirement of cytoskeletal interaction for the maintenance of adhesive properties does not seem to be absolute. For instance, T-cadherin, which naturally lacks the cytoplasmic and transmembrane domains, is still able to promote homotypic cell to cell aggregation (20).A recently discovered member of the cadherin family, vascular endothelial cadherin (VE-cadherin), 1 also known as cadherin-5 or 7B4 (8, 21), was found to be selectively expressed in endothelial cells of about all types of vessels (21). This molecule plays a role in the organization of lateral endothelial junctions (22) and in the control of permeability properties of vascular endothel...

Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-α, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-α mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.

In vivo, intact endothelium presents a low turnover rate, however, when junctions are disrupted cells gain the capacity to migrate and proliferate. This capacity is then lost when cell to cell contacts are reorganized (4).Endothelial cell junction components are therefore good candidates for transferring migration and growth inhibitory signals. Previous work (5) showed that protein membrane extracts from confluent endothelial cells were able to inhibit the growth of sparse endothelium but not of other cell types, suggesting the existence of membrane associated endothelial growth inhibitory proteins.It was previously found that endothelial cells express a cell specific member of the cadherin family (cadherin-5 or vascular endothelial cadherin [VE-cadherin]) 1 (6)(7)(8). This molecule is so far the only cadherin consistently organized at interendothelial adherence junctions (8-10). VE-cadherin is a constitutive component of all types of endothelia (8). As the other members of the family (11-15), VE-cadherin has adhesive properties and mediates homotypic cell adhesion (16). The intracellular domain interacts with cytoplasmic proteins called catenins (17) that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton (18,19).In other types of tissues, cadherins can act as tumor suppressors. Reduced cadherin expression and/or activity is associated with enhanced tumor cell invasive potential and loss of differentiated characteristics (11)(12)(13)(14)(15)(18)(19)(20)(21)(22)(23). In agreement with these observations, VE-cadherin expression was found to be strongly reduced in angiosarcomas (24).In this paper we investigated the effect of VE-cadherin on cell growth. The results reported show that VE-cadherin can indeed transfer growth negative signals to the cells. This molecule can therefore contribute to density dependent inhibition of endothelial cell growth. MethodsCells. Human endothelial cells from umbilical vein (HUVEC) were isolated and cultured in M199 and 20% NCS (newborn calf serum) as previously described (8). Chinese hamster ovary (CHO) cells and mouse connective tissue fibroblast L929 cells were obtained from the American Tissue Type Collection and cultured in DMEM with 10% FCS (both from GIBCO, Life Technologies, Paisley, U.K.) (16). Full length VE-cadherin cDNA was cloned from human endothelial cells and inserted into pECE eukaryotic expression vector. CHO and L929 cells were cotransfected with pECE-VE-cadherin construct and pSV 2 neo plasmids by calcium phosphate precipitation as described (16). Control cells were transfected with empty pECE and pSV 2 neo plasmids, selected, cloned, and cultured as VE-cadherin transfectants (16). VE-cadherin expression in the clones used was comparable with that of HUVEC by Western and Northern blot analysis (16).For transfection of CHO cells with truncated VE-cadherin, full length VE-cadherin cDNA cloned in pBluescript vector (16)

An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By immunohistochemistry, murine VE-cadherin protein was detected at cell-to-cell junctions of endothelial cells. Overall, these data demonstrate that VE-cadherin is an early, constitutive, and specific marker of endothelial cells. This distinguishes this molecule from other cadherins and suggests that its expression is associated with the early assembly of vascular structures.

The presence in plasma of an electronegative LDL subfraction [LDL(-)] cytotoxic for endothelial cells (ECs) has been reported. We studied the effect of LDL(-) on the release by ECs of molecules implicated in leukocyte recruitment [interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1)] and in the plasminogen activator inhibitor-1 (PAI-1). LDL(-), isolated by anion-exchange chromatography, differed from nonelectronegative LDL [LDL(+)] in its higher triglyceride, nonesterified fatty acid, apoprotein E and apoprotein C-III, and sialic acid contents. No evidence of extensive oxidation was found in LDL(-); its antioxidant and thiobarbituric acid-reactive substances contents were similar to those of LDL(+). However, conjugated dienes were increased in LDL(-), which suggests that mild oxidation might affect these particles. LDL(-) increased, in a concentration-dependent manner, the release of IL-8 and MCP-1 by ECs and was a stronger inductor of both chemokines than oxidized LDL (oxLDL) or LDL(+). PAI-1 release increased slightly in ECs incubated with both LDL(-) and oxLDL but not with LDL(+). However, no cytotoxic effects of LDL(-) were observed on ECs. Actinomycin D inhibited the release of IL-8 and MCP-1 induced by LDL(-) and oxLDL by up to 80%, indicating that their production is mediated by protein synthesis. Incubation of ECs with N:-acetyl cysteine inhibited production of IL-8 and MCP-1 induced by LDL(-) and oxLDL by >50%. The free radical scavenger butylated hydroxytoluene slightly inhibited the effect of oxLDL but did not modify the effect of LDL(-). An antagonist (BN-50730) of the platelet-activating factor receptor inhibited production of both chemokines by LDL(-) and oxLDL in a concentration-dependent manner. Our results indicate that LDL(-) shows proinflammatory activity on ECs and may contribute to early atherosclerotic events.

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