Polymorphism in the tissue factor region is associated with basal but not endotoxin-induced tissue factor-mRNA levels in leukocytes.

OBJECTIVE: Tissue factor (TF) plays a central role during disseminated intravascular coagulation (DIC) in sepsis. We hypothesized that a frequent D/I polymorphism, at nucleotide position -1208 in the promoter region, could influence TF-mRNA and downstream coagulation. METHODS: Basal- and lipopolysaccharide (LPS)-induced TF-mRNA expression, microparticle-associated TF-procoagulant activity and coagulation were determined in healthy men (n = 74) before and after endotoxin (LPS) infusion (2 ng kg(-1)). Basal values of TF-mRNA ranged between 34 and > 37.5 cycles. RESULTS: Baseline TF-mRNA levels significantly differed between genotypes: I/I carriers had almost 2-fold higher TF-mRNA levels compared to D/D carriers at baseline (P < 0.01). In accordance, higher levels of microparticle-associated TF-procoagulant activity could be seen in I/I carriers. However, the genotype did not affect basal or LPS-induced levels of prothrombin fragment F1+2, D-dimer or cytokines including tumor necrosis factor and interleukin-6. CONCLUSION: The TF-1208 polymorphism is functional in that it regulates basal TF-mRNA in circulating monocytes and circulating microparticle-associated TF-procoagulant activity in vivo, but does not influence the relative increase in TF-mRNA or coagulation activation during low-grade endotoxemia.

Cdc25A phosphatase suppresses apoptosis induced by serum deprivation.

The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.

Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherens junction functions.

Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.

Stringent endocrinological testing reveals subnormal growth hormone secretion in some patients with fibromyalgia syndrome but rarely severe growth hormone deficiency.

OBJECTIVE: Several reports suggest that growth hormone (GH) deficiency may be a pathogenic factor in fibromyalgia syndrome (FM). This hypothesis has never been adequately examined. METHODS: We measured serum GH concentration after insulin induced hypoglycemia in subjects with FM. GH secretion in subjects with a maximal GH increase < 10 ng/ml after hypoglycemia was assessed by additional arginine stimulation. RESULTS: In one of 56 subjects tested, GH remained below 3 ng/ml in both tests, satisfying the criteria for adult GH deficiency. Thirty-two subjects (67%) had a maximal GH > 10 ng/ml. We retrospectively found an inverse correlation between low density lipoprotein levels and maximal GH concentration in a subgroup of patients. CONCLUSION: Severe GH deficiency is not a significant pathogenic factor in most patients with FM. We observed an impaired reactivity of the somatotropic axis in one-third of patients with FM, in keeping with a functional alteration of the hypothalamus.

Endothelial adherens junctions.

The principle of the molecular organization of adherens junctions follows a uniform pattern, which is found in epithelial, muscular, neuroneal as well as in endothelial cells and is highly conserved among species. Transmembrane molecules of the cadherin family link to catenins, which anchor the adhesion plaque to the cytoskeleton. The kind of cadherin used in adherens junctions is cell-type specific, vascular endothelial (VE)-cadherin is specific for endothelial cells. The assembly and disassembly of the cadherin/catenin complex is dynamic and regulated by growth factors. The functional status of adherens junctions controls endothelial cell-to-cell adhesion, cell scattering, vessel morphogenesis and has intracellular signaling properties, thereby playing an important role in vasculogenesis and angiogenesis.

Vascular-endothelial cadherin (CD144)- but not PECAM-1 (CD31)-based cell-to-cell contacts convey the maintenance of a quiescent endothelial monolayer.

BACKGROUND: In vivo, all blood vessels are lined by a single layer of flattened noncycling endothelial cells. We tested the hypothesis that the maintenance of such a quiescent endothelial monolayer depends on homotypic contacts between not yet defined growth-inhibitory molecules located at interendothelial junctions. METHODS: ECV304 cells, which lack endogenous vascular endothelial cadherin (VE cadherin) or CD31 expression, were transfected with cDNA encoding for the respective proteins or with the empty vector. RESULTS: In VE cadherin transfectants, beta-catenin was targeted to junctional regions and the F-actin-based cytoskeleton formed parallel bundles reaching from one cell border to the other. In contrast, in CD31 transfectants and in empty vector cells, beta-catenin was dispersed throughout the cytoplasm, and F-actin formed short, plump and criss-cross bundles. On a two-dimensional plastic matrix, both, VE cadherin and CD31 transfectants formed clusters of polygonal cells, whereas in three-dimensional gels, only VE cadherin cells were able to form tubes. Empty vector cells grew in a fibroblast-like pattern and neither formed clusters nor tubes. Most importantly, whereas CD31 and empty vector cells grew on top of each other, formed polylayers and maintained cycling even after reaching confluence, VE cadherin cells strictly maintained a single layer of flattened cells and the numbers of cycling cells dramatically dropped after reaching a continuous monolayer. CONCLUSION: The insertion of VE cadherin into ECV304 cells produces a cell type which mimics endothelial growth characteristics seen in vivo.