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Biomedical subjects

G Camussi

Publications and source records attributed to G Camussi.

At least 91 records · Page 5Linked to original sources
PubMed1995

Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promotes in vivo angiogenesis by a heparin-dependent mechanism.

The aim of the present study was to investigate the angiogenic properties of platelet-activating factor (PAF). In vitro PAF was shown to induce a dose-dependent migration of human endothelial cells (EC) across the polycarbonate filters in Boyden's chambers. In contrast, D-PAF, the biologically inactive enantiomer, and Lyso-PAF did not stimulate a significant migration of EC. This effect of PAF was not associated with a proliferative response of EC to this mediator. Moreover, the ability of PAF to stimulate the migration of EC was independent of the presence of heparin in the medium. WEB 2170, a specific PAF receptor antagonist, prevented the migration of EC induced by PAF, thus suggesting a receptor-dependent stimulation. The expression of PAF receptor gene by EC was confirmed by reverse transcriptase-PCR and Southern blot analysis. The in vivo angiogenic effect of PAF was studied in mice using a model in which Matrigel was used for the delivery of mediators. PAF induced a dose-dependent angiogenic response, which at pharmacologic concentrations (1-5 microM) did not require heparin, but at physiologic concentrations (5-50 nM) required the presence of heparin at doses that were not angiogenic per se. The angiogenesis induced by 50 nM PAF was, indeed, inhibited both by protamine and by the PAF receptor antagonist WEB 2170. The angiogenic effect of D-PAF and Lyso-PAF was not significant. Neutralizing Abs to basic fibroblast growth factor induced a slight but not statistically significant reduction of the angiogenesis induced by PAF.

Animals↗
PubMed1995

Platelet-activating factor produced by endothelial cells. A molecule with autocrine and paracrine properties.

Endothelial cells (EC) participate in microenvironment homeostasis by regulating the trafficking of cells and molecules from the bloodstream to the tissues. The 'area codes' used to control the trafficking are adhesion molecules, receptors which pick up external signals, and mediators of cell-to-cell communication. Platelet-activating factor (PAF) is a mediator that belongs to this class of 'area-code' molecules. PAF is an acetylated derivative of phosphatidylcholine which is produced by EC and may act in an autocrine manner. Several stimuli may induce the synthesis of PAF by EC with different time courses. Thrombin, elastase and tumor necrosis factor (TNF) are prototypic molecules inducing very-early, early and delayed PAF synthesis, respectively. These stimuli activate the 'remodelling pathway' which requires the activation of phospholipase A2 and acetyl CoA:1-alkyl-2-lyso-sn-glycero-3-phosphate acetyltransferase. The effects of very-early and early stimuli are mediated by a direct stimulation of this pathway, whereas the action of TNF is mediated by the new synthesis of a serine protease. Stimulated EC produce PAF molecules with both an ether and an ester bond at the sn-1 position, but the former is predominant. The PAF produced is partially exposed on the external membranes and participates in adhesion or migration of neutrophils. PAF released can also stimulate EC themselves, by interacting with a specific receptor. PAF stimulates the migration and the shape change of EC by activating calcium influx and several serine/threonine and tyrosine kinases which regulate the cytoskeleton.

Cell Adhesion↗
PubMed1995

Removal of platelet-activating factor in experimental continuous arteriovenous hemofiltration.

OBJECTIVE: There is a positive correlation between the amount of ultrafiltration and the improved survival rate of patients with ischemia or sepsis-induced acute renal failure. Continuous arteriovenous hemofiltration (CAVH) removes vasoactive substances with a molecular weight of < 1000 daltons. This study evaluated the removal of platelet-activating factor, a lipid mediator of endotoxic shock, by CAVH with respect to kinetics, adsorption, and ultrafiltration. DESIGN: Prospective laboratory study. SUBJECTS: Normal human subjects. INTERVENTIONS: Radioactive [3H] or biologically active platelet-activating factor was added to whole blood or washed blood resuspended in Tris-buffered (pH 7.2) physiologic saline with 4% human serum albumin or plasma. Whole or washed blood cells or plasma were recirculated at 100 mL/min through polysulfone hemofilters for 120 mins with ultrafiltration (condition A), without ultrafiltration (condition B), or in a static condition (condition C). Concentrations of albumin, total protein, and radioactive or biologically active platelet-activating factor in samples obtained from the blood and ultrafiltrate compartment were determined. MEASUREMENTS: Biologically active platelet-activating factor was quantified on washed rabbit platelets and results were expressed in ng/mL over a calibration curve obtained with synthetic platelet-activating factor. MAIN RESULTS: [3H]-platelet-activating factor added to recirculated whole blood was ultrafiltered (percent of ultrafiltered platelet-activating factor/min: 0.48 +/- 0.02 [SD]; total platelet-activating factor removed in 120 mins: 15.52%; condition A) at significantly (p < .001) higher amounts than when added to washed blood cells (percent of ultrafiltered platelet-activating factor removed/min: 0.195 +/- 0.06; total platelet-activating factor removed in 120 mins: 7.46%). The highest amounts of [3H]-platelet-activating factor were bound to polysulfone membranes after recirculation with whole blood (44.5 +/- 12.2%) than with washed blood (1.1 +/- 0.3%) or plasma (11.9 +/- 0.7%). Biologically active platelet-activating factor concentrations significantly decreased in both conditions A and B (maximal decrease at 120 mins: 63% and 59%, respectively). No significant reduction could be observed in condition C. CONCLUSIONS: These studies provide experimental evidence for the prompt, efficient removal of platelet-activating factor in CAVH and provide a possible rationale for the beneficial effect of this therapy in the development of multiple organ failure in sepsis.

Adsorption↗
PubMed1995

In vivo activation of met tyrosine kinase by heterodimeric hepatocyte growth factor molecule promotes angiogenesis.

Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for epithelial cells. The factor is a 90-kD heterodimer composed of an alpha chain containing four kringle motifs and a beta chain showing structural homologies with serine proteases. It is, however, devoid of enzymatic activity. Recently, it has been reported that HGF activates migration and proliferation of endothelial cells and is angiogenic. In this article we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mutants, and (2) the characteristics of the angiogenic response to HGF in an experimental model system of implanted reconstituted basement membrane (Matrigel). Two groups of mutants were made and used in vitro and in vivo: one with deletions of kringle domains and one with substitution at the cleavage site of the HGF precursor. In vitro, HGF variants containing only the first two (HGF-NK2) or the first three kringles (HGF-NK3) of the alpha chain did not induce proliferation of endothelial cells even if used at concentration 160-fold higher than that optimal for HGF (0.05 nmol/L). High concentrations of these mutants (4 to 8 nmol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gln 489 in the HGF precursor generated an uncleavable single-chain factor, unable to induce either endothelial cell migration or proliferation. In vivo, HGF induced a dose-dependent angiogenic response, which was enhanced by heparin.

Animals↗
PubMed1995

Adherence of human monocytes to haemodialysis membranes: LFA 1 (CD11a/CD18) CR1 (CD35) and CR3 (CD11b/CD18) triggering promotes the biosynthesis of platelet-activating factor and adherence.

BACKGROUND: Platelet-activating factor is a mediator of inflammation involved in the blood-membrane interaction. We report that selective stimulation of complement receptors (CR1 and CR3) triggers PAF synthesis and monocyte adherence to complement-activating membranes. METHODS: The synthesis of PAF was studied after stimulation of normal human adherent monocytes with F(ab)2 and Fab fragments of monoclonal antibodies specific to CR1 and CR3. CD11a, CD11b, CD18, and CD35 was studied by flow cytometry on neutrophils and monocytes. The molecular species of PAF from stimulated monocytes were identified by reverse-phase high-performance liquid chromatography coupled with mass spectrometry. RESULTS: Anti-CR1 and anti-CR3 monoclonal antibodies induced a dose-dependent C-16 but not C-18 PAF production. The latter occurred also with monovalent Fab fragments of both anti-CR1 and anti-CR3 monoclonal antibodies, that were not internalized as seen by immunofluorescence. Adherence of monocytes to Cuprophan membranes was markedly higher (P < 0.01) in membranes pretreated with fresh than with heat-inactivated normal plasma. However, the high adherence to fresh plasma-treated membranes was completely abrogated by coincubating the cells with Web 2170, a specific PAF receptor antagonist. This was not due to downregulation of adhesion molecules expression on leukocytes. CONCLUSIONS: These studies implicate a crucial role of PAF in blood interaction with haemodialysis membranes that fix complement activated products.

Antibodies, Monoclonal↗
PubMed1994

Role of platelet activating factor in acute pancreatitis induced by lipopolysaccharides in rabbits.

In the present study we demonstrated that a single injection of endotoxin (lipopolysaccharides, E. Coli 0111-B4) into the superior pancreaticoduodenal artery of rabbits induced a dose-dependent acute necrotizing pancreatitis. The lesions observed by light microscopy were significant for 10 micrograms lipopolysaccharides and were maximal for 20 micrograms. After 24 h the main findings were edema, acinar cell vacuolisation, polymorphonuclear neutrophil infiltration and tissue necrosis. The pancreatic lesions developed strictly in the area supplied by the artery injected with lipopolysaccharides, without significant intestinal involvement. Since platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine, PAF; 50-500 ng), a phospholipid mediator of endotoxin-induced inflammation and shock, was previously shown to cause an acute necrotizing pancreatitis in rabbits, the role of PAF in the development of acute pancreatitis induced by lipopolysaccharides was studied by evaluating: (1) the synergism between doses of lipopolysaccharides (5-10 micrograms), which produced a mild tissue injury, and doses of PAF (10 ng) not producing, per se, any significant injury, and (2) the effect of three structurally unrelated PAF receptor antagonists. The results obtained demonstrated that 10 ng of PAF significantly potentiated pancreatic tissue damage induced by 10 micrograms of lipopolysaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis.

Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug.

Animals↗
PubMed1994

Human endothelial cells are targets for platelet-activating factor (PAF). Activation of alpha and beta protein kinase C isozymes in endothelial cells stimulated by PAF.

We evaluated the role of the protein kinase C (PKC) and its isozymes in the activation of human endothelial cells (EC) stimulated by platelet-activating factor (PAF). Exposure of confluent EC to PAF resulted in a rapid and concentration-dependent redistribution of PKC from cytosol to plasma-membrane, rearrangement of cytoskeleton (i.e. decrease in F-actin content and redistribution of vinculin), and finally increase in the transendothelial flux of 125I-albumin. Stimulation of EC with oleylacetylglycerol or phorbol 12-myristate 13-acetate induced the modification of the cytoskeletal structures and the increase of 125I-albumin clearance. Inhibitors of PKC prevented the effects induced by PAF on the cytoskeleton and on the barrier function of the EC monolayer. Confluent EC expressed only alpha, beta, and epsilon PKC isoforms. Biochemical and immunochemical analysis showed that the time course of the PKC isozymes translocation from cytosol to the membrane fraction of EC stimulated by PAF was different: beta isoform was redistributed more quickly than alpha isoform. PAF did not induce translocation of PKC epsilon. These results suggest that activation of PKC alpha and beta is an important signal transduction pathway by which PAF activates endothelial monolayer and modify its function of barrier to macromolecules.

Actins↗
PubMed1994

Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-alpha or interleukin-1 alpha.

It has been shown that production of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha requires the synthesis of new proteins and is regulated by anti-proteinases. Here, we demonstrate that TNF-alpha and IL-1 alpha induce the expression by EC of a 34-kDa diisopropyl fluorophosphate-binding protein immunoprecipitated by an anti-human elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N-methoxysuccinyl- Ala-Ala-Pro-Val p-anilide, which is specific for elastase. The generation of this elastase-like protein seems to be important for the synthesis of PAF induced by TNF-alpha and IL-1 alpha, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti-human elastase antibody prevent the synthesis of PAF and the activation of 1-O-alkyl-2-lyso-glycerophosphocholine acetyl-CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine-activated EC, elicits a rapid synthesis of PAF by EC. High-performance liquid chromatography-tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF-alpha, IL-1 alpha or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF-alpha and IL-1 alpha stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.

Acetyltransferases↗
PubMed1994

Alternative pathway activation of complement by cultured human proximal tubular epithelial cells.

Human proximal tubular epithelial cells (PTEC) incubated with normal human serum (NHS) were found to fix on their surface C3, properdin, terminal complement components and C5b-9 MAC neoantigen, but not C1q and C4, by immunofluorescence. Complement fixation was abrogated if PTEC were incubated with EDTA-treated NHS or C3-deficient human serum, but not with Mg EGTA-treated NHS or C1q-deficient human serum, showing the prevalent activation of the alternative pathway of complement. This event was followed by marked cytoskeleton alterations with disruption of the actin cortical network, redistribution of actin throughout the cytoplasm and formation of blebs, and by cell cytolysis. In addition, superoxide anion and hydrogen peroxide production and chemiluminescence response were detected in consequence of MAC insertion on PTEC plasma membrane. The dependency on MAC of the observed biological effects of complement fixation on PTEC surface was shown by using sera selectively deficient of terminal components of complement (C6 or C8), and therefore unable to form the C5b-9 MAC, and by restoring the ability to form MAC after addition of purified C6 or C8. The possible pathogenetic relevance of these observations in tubulointerstitial injury occurring in patients with complementuria due to non-selective proteinuria, is discussed.

Blood↗
PubMed1994

Platelet-activating factor-induced endothelial cell expression of adhesion molecules and modulation of surface glycocalyx, evaluated by electron spectroscopy chemical analysis.

PAF synthesized by or added to the endothelial cell monolayer has as first target the endothelium itself, inducing the expression of GMP-140 on the cell surface and qualitative alterations in the composition of glycocalyx. As seen by ESCA, sulfated chemical groups were significantly reduced after PAF stimulation. This reduction depended on selective loss of sulfated proteoglycans that were released into the supernatant. The alteration of glycocalyx may favor functional exposure of GMP-140 and of PAF on the surface of endothelial cells and may reduce charge repulsive forces between cells. PAF associated with endothelial cell surface has as second target PMNs and stimulates in cooperation with GMP-140, the functional activation of PMN CD11-CD18 adhesion molecules.

Antibodies, Monoclonal↗
PubMed1994

Role of platelet-activating factor (PAF) in oxygen radical-induced cardiac dysfunction.

Several studies established the role of oxygen radicals in cardiac alterations occurring during ischemia and reperfusion. Recently, platelet-activating factor (PAF), a phospholipid mediator of inflammation, was also implicated in ischemia-reperfusion injury. The present study was performed to evaluate whether biosynthesis of PAF may mediate the mechanical and electrical alterations induced by perfusion with dihydroxyfumaric acid (DHF; 1 mM), a free radical-generating compound, in guinea pig isolated perfused heart and isolated atrium. The results obtained indicate that DHF induces an intracoronary production of PAF (DHF-perfused hearts = 43.1 +/- 3.9 pg PAF; saline-perfused control hearts = PAF undetectable) and electrical and mechanical alterations in both isolated heart and atrium. These effects were shown to be dependent on superoxide (O2-) generation, because they were completely prevented by superoxide dismutase (10 mM), and were absent when DHF's ability to produce O2- was exhausted in solution. The role of PAF in mediating oxygen radical-induced electrical and mechanical alterations was established by pretreatment of cardiac preparations with WEB 2170 (0.1-10 microM), a specific PAF-receptor antagonist devoid of any direct antioxidant activity. At the concentration of 3 microM, WEB 2170 abrogated almost completely all the cardiac effect of DHF. These results suggest that PAF may act as secondary mediator of oxygen radicals in the heart.

Animals↗
PubMed1993

Development of a high-performance liquid chromatographic-mass spectrometric technique, with an ionspray interface, for the determination of platelet-activating factor (PAF) and lyso-PAF in biological samples.

An HPLC-mass spectrometric technique with an ionspray interface was developed for the determination of platelet-activating factor (PAF) and PAF-related compounds in biological samples. HPLC separations were performed using a reversed-phase column. The mass spectra showed intense [M + H]+ ions. Collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the phosphorylcholine group. By selective-ion monitoring, a detection limit of 0.3 ng was obtained for all molecules; by multiple reaction monitoring, the same sensitivity was achieved for PAF whereas for lyso-PAF the limit was 3 ng. Finally, PAF was comparatively determined by bioassay and HPLC-MS after extraction from the cell pellets and the supernatants of human polymorphonuclear neutrophils unstimulated or stimulated with opsonized zymosan. The good correlation observed between these techniques indicated the reliability of HPLC-MS for biochemical studies on PAF and PAF-related molecules.

Chromatography, High Pressure Liquid↗
PubMed1993

Outer-membrane porins from gram-negative bacteria stimulate platelet-activating-factor biosynthesis by cultured human endothelial cells.

Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.

Acetyltransferases↗