Chlamydia species infect human vascular endothelial cells and induce procoagulant activity.

BACKGROUND: Chlamydia pneumoniae infections have been linked with myocardial infarction, stroke, and the development of atherosclerosis by epidemiologic studies, immunohistochemical studies, and electron microscopic studies. The mechanisms underlying this association are unknown. METHODS: Using cultured human venous endothelial cells, we investigated whether C pneumoniae, C trachomatis (types H and L2/434/BU) could infect these cells. The ability of infected cells to express procoagulant (tissue factor) activity was also measured using clotting and chromogenic substrate assays. Adhesion of platelets to chlamydia-infected cells was also quantitated. RESULTS: We found that C pneumoniae, C trachomatis type H, and C trachomatis L2/434/BU could infect cultured human umbilical vein endothelial cells and stimulate a 4-fold increase in expression of tissue factor, which reached a peak 18 hours postinfection. Tissue factor expression was enhanced even in the presence of tetracycline, suggesting that the chlamydial factor responsible for stimulating synthesis of endothelial cell tissue factor was preformed. Platelet adhesion was significantly enhanced when endothelial cells were infected by chlamydia species. CONCLUSIONS: These in vitro studies suggest possible pathogenic mechanisms that may explain the association of thrombotic events with C pneumoniae infection, including pathologically enhanced production of tissue factor by human endothelial cells and enhanced focal platelet deposition.

Truncated trkB receptors on nonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro.

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These results provide strong support for the hypothesis that the up-regulation of trkB-T1 receptors on astrocytes following CNS lesions enhances the sequestration of the trkB ligands, BDNF and NT- 4/5, at the site of reactive gliosis and, thus, contributes to the inhibition of CNS axonal regeneration from neurons expressing trkB-kinase receptors by removing their ligands from the extracellular environment.

Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain.

The neurotrophins brain-derived neurotrophic factor (BDNF) and NT-4/5 exert their trophic effects on the nervous system via signaling through trkB receptors. These receptors occur as splice variants of the trkB gene that encodes a full-length receptor containing the signal transducing tyrosine kinase domain as well as truncated forms lacking this domain. Because the importance of the trkB isoforms for development and maturation of the nervous system is unknown, we have examined the expression of trkB receptor isoforms during development of the rat forebrain using 1) a sensitive ribonuclease protection assay to distinguish full-length and truncated trkB transcripts, 2) western blot analysis to characterize developmental changes in trkB proteins, and 3) immunohistochemistry to determine the cellular localization of trkB receptors. In the rat forebrain, adult mRNA levels for full-length trkB are reached by birth, whereas truncated trkB message does not peak until postnatal days 10-15. Western blot analysis indicates that full-length trkB protein is the major form during early development, whereas truncated trkB protein predominates in all forebrain regions of late postnatal and adult rats. These data also suggest that the glycosylation state of these receptors changes during postnatal maturation. TrkB immunoreactivity is present predominately within neurons, where it is localized to axons, cell soma, and dendrites. Strong dendritic immunostaining is particularly evident in certain neuronal populations, such as pyramidal neurons in the hippocampus and in layer V of the neocortex. The dendritic localization of trkB receptors supports the hypothesis that dendrites, as well as axons, are important sites for neurotrophin actions in the central nervous system.

Homocysteine, a risk factor for premature vascular disease and thrombosis, induces tissue factor activity in endothelial cells.

Elevated blood levels of homocysteine represent an independent risk factor for premature arterial vascular disease and thrombosis. We investigated whether homocysteine could induce tissue factor (TF) procoagulant activity in cultured human endothelial cells. Homocysteine increased cellular TF activity in a time- and concentration-dependent manner. Low concentrations of homocysteine (0.1 to 0.6 mmol/L), similar to those found in the blood of patients with homocystinuria, enhanced TF activity by 25% to 100%. Other sulfur-containing amino acids (cystine, homocystine, cysteine, and methionine) induced less TF activity than did homocysteine; however, beta-mercaptoethanol and dithiothreitol were more effective than homocysteine in increasing TF activity. Preincubation of homocysteine with a sulfhydryl inhibitor such as N-ethylmaleimide prevented homocysteine induction of TF activity. A quantitative polymerase chain reaction method indicated that homocysteine increased TF mRNA in endothelial cells. These results indicate that an atherogenic amino acid, homocysteine, can initiate coagulation by the TF pathway through a mechanism involving the free thiol group of the amino acid and by TF gene transcription. These data support the hypothesis that perturbation of vascular coagulant mechanisms may contribute to the thrombotic tendency seen in patients with homocystinuria.