Electronegative LDL from normolipemic subjects induces IL-8 and monocyte chemotactic protein secretion by human endothelial cells.

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.

Inhibition of cultured cell growth by vascular endothelial cadherin (cadherin-5/VE-cadherin).

Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.

Effect of advanced glycosylation end products on the induction of nitric oxide synthase in murine macrophages.

We studied the role of advanced glycosylation end products on the induction of nitric oxide synthase in peritoneal mouse macrophages previously exposed to modified BSA. A dose-dependent increment in the nitric oxide production induced by LPS and IFN-gamma was observed when cell cultures were pretreated with modified BSA for 48 hours. In addition, the up regulation of nitric oxide production was also time-dependent, being maximal at 24-48 hours. Experiments carried out in the presence of neutralizing antibodies to IL-1 and TNF-alpha, suggested that up regulation was not due to the capacity of modified BSA to induce both proinflammatory signals. The up regulation of nitric oxide production was paralleled with an increase in iNOS mRNA.

Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.

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.

Nitric oxide modulates interleukin-2-induced proliferation in CTLL-2 cells.

The role of the L-arginine-nitric oxide metabolic pathway was explored for interleukin-2-induced proliferation in the cytotoxic T lymphocyte clone CTLL-2. Specific inhibition of nitric oxide synthase significantly diminished, in a concentration-dependent manner, (3)H-thymidine uptake of CTLL-2 cells in response to different concentrations of interleukin 2. Withdrawal of L-arginine from culture medium resulted as potent as the higher inhibition obtained when blocking nitric oxide synthase with L-arginine analogues. Furthermore, intermedial concentrations of Larginine and exogenous nitric oxide donors were found for achieving optimal IL2-induced proliferation of CTLL-2. These findings prompted us to suggest that intra- and/or inter-cellular nitric oxide signalling may contribute to the modulation of the IL2 mitogenic effect upon cytotoxic T lymphocytes.

Catenin-dependent and -independent functions of vascular endothelial cadherin.

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 VE-cadherin 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.

Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.

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.

The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin).

In this paper we report that the assembly of interendothelial junctions containing the cell type-specific 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, alpha-catenin, and beta-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, alpha-catenin, beta-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, alpha-catenin, and beta-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, alpha-catenin, and beta-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascant 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, alpha-catenin, and beta-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VE-cadherin/alpha-catenin/beta-catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.

Lobenzarit disodium inhibits the constitutive NO-cGMP metabolic pathways. Possible involvement as an immunomodulatory drug.

Lobenzarit disodtulIl (CCA) is a novel immunomodulatory drug useful in the treatment of chronic inflammations. Its principal mechanism of action seems to be through enhancing the T suppressor/T helper lymphocyte ratio. However, the molecular basis for these actions remains unclear. In this study it was found that CCA inhibits the production of guanosine 3',5'-cyclic monophosphate almost completely when present in concentrations of 1 mM. Further results demonstrated that such inhibition could also be explained by interference in constitutive nitric oxide generation. In addition to previous findings, more insight into the molecular mechanism of action of CCA is provided.

Leukocyte-endothelial cell adhesive receptors.

The first step in leukocyte localization at inflammatory foci is their adhesion to the endothelial surface. This is a complex process mediated by several adhesive molecules expressed both on the leukocyte and endothelial membrane. In the early phases of inflammation, leukocytes transiently adhere to the vessel wall in a process termed "rolling". Rolling of leukocytes is mediated by a family of adhesive molecules called selectins, expressed both on the leukocyte and endothelial surface. Other adhesive molecules and chemotactic agents act in a complementary way, with selectins stabilizing polymorphonuclear cell adhesion and mediating their transendothelial migration into the inflamed foci. In particular, leukocyte beta 2 integrins present on the leukocyte membrane, binding to two adhesive immunoglobulins (ICAM-1 and ICAM-2) on the endothelial surface. Monocytes and lymphocytes also express the integrin VLA-4 (alpha 4 beta 1) which is the ligand of the immunoglobulin VCAM-1 on endothelial cells. It is still unknown how leukocytes can migrate through the inter-endothelial junctions. An interesting possibility is that leukocyte adhesion to endothelial cells could trigger intracellular signals that in turn can mediate junction disassembly. An understanding of the molecular mechanisms at the basis of leukocyte adhesion to the vessel wall and of their infiltration into the inflamed area could help to develop specific antagonists and a more targeted therapy for inflammatory diseases.

Endothelial cell-to-cell junctions. Structural characteristics and functional role in the regulation of vascular permeability and leukocyte extravasation.

Endothelial monolayer forms the main barrier to the passage of macromolecules and circulating cells from blood to tissues. This property is regulated by intercellular junctions. These are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic cytoskeletal proteins. Endothelial junctions vary in number and organization along the vascular tree. Some transmembrane components of endothelial junctions have recently been identified. One is specifically expressed by endothelial cells (cadherin-5) while others (such as PECAM-1 and integrins) are also present in other cell types. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is possible that inflammatory agents increase permeability by binding to specific receptors on the endothelial membrane. This would lead to the generation of intracellular signals causing cytoskeletal reorganization and opening of interendothelial gaps. This general sequence of events, however, seems to follow specific routes for different stimuli. In fact, permeability-increasing agents differ in the type of intracellular second messenger they trigger, for the time course of their effect, and for their specificity for the endothelium of different vascular districts. Endothelial junctions also regulate leukocyte extravasation. Endothelial cells actively contribute to this process by expressing adhesive molecules on their surface and by releasing chemotactic cytokines. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial junctions occurs. The mechanism by which this takes place is unknown but it might resemble that triggered by soluble inflammatory mediators.

Role of nitric oxide pathway in the protection against lethal endotoxemia afforded by low doses of lipopolysaccharide.

Survival after lipopolysaccharide challenge (LD80, 20 mg.kg-1, i.p.) was significantly enhanced by previous treatment with a microdose of LPS (50 micrograms.kg-1, i.v.). When NG-monomethyl-L-arginine, a specific inhibitor of the formation of nitric oxide from L-arginine, was given 30 minutes before microdose, survival was significantly reduced. When we monitored the serum Tumor Necrosis Factor (TNF) levels in both groups a significant reduction of TNF level after the microdose was observed in mice previously treated with L-NMMA. The ability of L-NNMA to reduce TNF release was dose dependent.