Recombinant activated protein C attenuates endothelial injury and inhibits procoagulant microparticles release in baboon heatstroke.

OBJECTIVE: We tested the hypothesis that the antithrombotic and cytoprotective effects of recombinant human activated protein C (rhAPC) protect baboons against the lethal effects of heatstroke. METHODS AND RESULTS: Fourteen anesthetized baboons assigned randomly to rhAPC (n=7) or control group (n=7) were heat-stressed in a prewarmed incubator at 44 to 47 degrees C until systolic blood pressure fell below 90 mm Hg, which signaled severe heatstroke. rhAPC was administered intravenously (24 microg/kg/h) for 12 hours at onset of heatstroke. Heat stress induced coagulation and fibrinolysis activation as evidenced by a significant increase from baseline levels in plasma levels of thrombin-antithrombin (TAT) complexes, tissue plasminogen activator, and D-dimer. Heat stress elicited cell activation/injury as assessed by the release of interleukin (IL)-6, soluble thrombomodulin, and procoagulant microparticles (MPs). rhAPC did not significantly reduce heatstroke-induced thrombin generation, and D-dimer and had no effect on fibrinolytic activity. In contrast, rhAPC infusion attenuated significantly the plasma rise of IL-6 and inhibited the release of soluble thrombomodulin and MPs as compared with control group. No difference in survival was observed between rhAPC-treated and control group. CONCLUSIONS: rhAPC given to heatstroke baboons provided cytoprotection, but had no effect on heatstroke-induced coagulation activation and fibrin formation. Inhibition of MPs by rhAPC suggested a novel mechanism of action for this protein.

Exaggerated apoptosis and NF-kappaB activation in pancreatic and tracheal cystic fibrosis cells.

The pathophysiologic mechanisms causing inflammation in cystic fibrosis (CF) remain obscure. The effects of proapoptotic agents on pancreatic and tracheal cell lines expressing wild-type CFTR (PANC-1 and NT-1, respectively) or the homozygous CFTRDeltaF508 mutation (CFPAC-1 and CFT-2, respectively) were assessed. An increased susceptibility to apoptosis was observed in CFPAC-1 and CFT-2 cells. Apoptosis was reduced by treatment with a pan-caspase inhibitor and by incubation at 27 degrees C, allowing recruitment of CFTR deltaF508 at the plasma membrane. Inhibition of CFTR function in wild-type cells induced an increase of apoptosis. Apoptosis in CFPAC-1, but not in CFT-2 cells, was associated with overexpression of the proinflammatory mediators interleukin-6 and interleukin-8. In CF cells, apoptosis was linked to NF-kappaB pathway activation. Conditioned medium from actinomycin D-treated CFPAC-1 cells produced an increase in apoptosis of wild-type cells, suggesting that proinflammatory mediators secreted by mutant cells promote apoptosis. This was confirmed through the induction of apoptosis in wild-type cells by exogenous interleukin-6 and interleukin-8. These results suggest that CFTR deltaF508 mutation, apoptosis, and activation of the NF-kappaB pathway contribute to the self-perpetuating inflammatory cycle, at least in pancreatic cells, and provide evidence that excessive apoptosis may account for the exaggerated proinflammatory response observed in CF patients.

Phosphatidylserine and signal transduction: who needs whom?

The plasma membrane, long considered a simple barrier between the extracellular and intracellular compartments, is now thought to play a pivotal role in many physiological processes that regulate the communication of cells with their environment. On one hand, the plasma membrane directly participates in intracellular signaling; on the other hand, changes in membrane structure contribute to the transcellular transfer of biological information. Among the membrane constituents, phosphatidylserine is a major actor implicated in these effects. Evidence now exists for a role for phosphatidylserine redistribution in modulating the activities of several membrane proteins during signaling in nonapoptotic T lymphocytes.

[Plasma membrane remodelling and cell stimulation]. / Remodelage de la membrane plasmique et stimulation cellulaire.

For a long time the plasma membrane has been considered as a simple barrier between the extracellular and intracellular milieu. Now, it is well accepted that it plays a pivotal role in many physiological processes allowing the communication of cells with their environment. On the one hand, the plasma membrane directly participates in intracellular signaling, on the other hand, changes in membrane structure contribute to the transcellular transfer of biological information. This review analyses the most recent features concerning the plasma membrane plasticity, with a special focus on the intracellular signaling pathways involved in the regulation of the loss of membrane phospholipid asymmetry during cell activation. The pathophysiologic consequences of microparticle/microvesicle shedding from membrane blebs are briefly exposed.

Membrane microvesicles as actors in the establishment of a favorable prostatic tumoral niche: a role for activated fibroblasts and CX3CL1-CX3CR1 axis.

Tumor microenvironment is enriched in plasma membrane microvesicles (MV) shed from all cell types that constitute the tumor mass, reflecting the antigenic profile of the cells they originate from. Fibroblasts and tumor cells mutually communicate within tumor microenvironment. Recent evidences suggest that tumor-derived MVs (TMV) exert a broad array of biological functions in cell-to-cell communication. To elucidate their role in cancer-to-fibroblast cell communication, TMV obtained from two prostate carcinoma cell lines with high and weak metastatic potential (PC3 and LnCaP, respectively) have been characterized. TMV exhibit matrix metalloproteinases (MMP) and extracellular MMP inducer at their surface, suggesting a role in extracellular matrix degradation. Moreover, TMV not only induce the activation of fibroblasts assessed through extracellular signal-regulated kinase 1/2 phosphorylation and MMP-9 up-regulation, increase motility and resistance to apoptosis but also promote MV shedding from activated fibroblasts able in turn to increase migration and invasion of highly metastatic PC3 cells but not LnCaP cells. PC3 cell chemotaxis seems, at least partially, dependent on membrane-bound CX3CL1/fractalkine ligand for chemokine receptor CX3CR1. The present results highlight a mechanism of mutual communication attributable not only to soluble factors but also to determinants harbored by MV, possibly contributing to the constitution of a favorable niche for cancer development.

Fluorescent biomembrane probe for ratiometric detection of apoptosis.

Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4'-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.

Membrane microparticles: two sides of the coin.

Microparticles are plasma membrane-derived vesicles shed from stimulated cells, in the broad sense of the term. Their presence is interpreted by proximal or remote cells in fundamental physiological processes including intercellular communication, hemostasis, and immunity. On the other hand, variations of their number or characteristics are frequently observed in pathophysiological situations.

Circulating cell-derived microparticles in Crohn's disease.

Procoagulant membrane microparticles can be released from activated or apoptotic cells in response to various environmental stimuli. The aim of this study was to investigate the presence of microparticles in Crohn's disease and to assess their variations after infliximab therapy. We compared the levels of circulating microparticles in 38 patients with Crohn's disease, 16 patients with ulcerative colitis, 7 patients with infectious colitis, and 17 control subjects. The evolution of microparticle levels was assessed after infliximab therapy in 13 patients with Crohn's disease. Circulating microparticle levels were elevated in patients with Crohn's disease (9.31+/-0.66 nmol/L phosphatidylserine equivalent [PS Eq]) or infectious colitis (10.71+/-0.92 nmol/L PS Eq) compared to patients with ulcerative colitis (5.75+/-0.59 nmol/L PS Eq) and control subjects (4.06+/-0.37 nmol/L PS Eq) (P = 0.001). Infliximab induced a significant diminution of the amounts of circulating microparticles, from 10.33+/-1.20 to 6.45+/-0.90 nmol/L PS Eq (P = 0.002). Generation of circulating microparticles occurs in Crohn's disease; infliximab induces significant diminution. Release of microparticles could be linked to the type of inflammatory response underlying Crohn's disease.