Effect of Robola and Cabernet Sauvignon extracts on platelet activating factor enzymes activity on U937 cells.

A number of studies support the anti-atherogenic effect of wine compounds. The scope of this study was to examine the effect of a red (Cabernet Sauvignon-CS) and a white (Robola-R) wine, as well as resveratrol and quercetin, on the platelet activating factor (PAF) biosynthetic enzymes, acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), and its main catabolic enzyme (PAF acetylhydrolase; PAF-AH), on U937 cells, in cell free and in intact cell experiments. In cell free experiments, phenolic compounds and wine extracts inhibited PAF biosynthetic enzymes, however in higher concentrations than intact cell experiments. In the latter cases, polar lipids of both wines inhibited in the same order of magnitude the action of lyso-PAF-AT and of PAF-CPT. The water fractions possessed a dual action, in lower concentrations they activated both enzymes, while in higher concentrations only inhibited PAF-CPT. All fractions either did not affect or slightly activated PAF-AH activity. In conclusion, wine compounds may exert their anti-inflammatory activity by reducing PAF levels through modulation of the PAF metabolic enzymes.

Effects of highly active antiretroviral therapy on platelet activating factor metabolism in naive HIV-infected patients: ii) study of the abacavir/lamivudine/efavirenz HAART regimen.

Human Immunodeficiency Virus (HIV)-infected patients are at increased risk for cardiovascular diseases partly due to chronic inflammation. Some antiretroviral drugs and Highly Active Anti-Retroviral Therapy (HAART) regimens seem to be related and amplify this increased risk, especially the ones containing abacavir. Platelet-Activating-Factor (PAF) is a potent inflammatory mediator that is implicated in both cardiovascular diseases and HIV-related manifestations. Our objective is to study the in vivo effect of the abacavir/lamivudine/efavirenz first-line HAART regimen on PAF metabolism in HIV-infected patients. The specific activities of PAF basic biosynthetic enzymes in leukocytes and platelets, PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), but also those of PAF-basic catabolic enzymes, PAF acetylhydrolase (PAF-AH) in leukocytes and platelets and Lipoprotein-associated-Phospholipase-A2 (LpPLA2) in plasma, were measured in blood samples of 10 asymptomatic naïve male HIV-infected patients just before and after 1, 3 and 6 months of treatment. CD4 cell counts, viral load and several biochemical markers were also measured in the same blood samples of these patients. The repeated ANOVA measures and the Pearson r criterion were used for studying statistical differences and correlations - partial correlations respectively. Even though viral load was decreased and CD4 cell counts were beneficially increased after treatment with the abacavir/lamivudine/efavirenz regimen, the main enzyme of the remodelling PAF-synthesis that is implicated in pro-atherogenic inflammatory procedures, Lyso-PAF-AT activity, was increased at 3 months of treatment in both leukocytes and platelets, while the main enzyme of PAF-degradation, PAF-AH, was increased as a response only in leukocytes at the 3rd month. Although the abacavir/lamivudine/efavirenz HAART regimen exhibits very efficient antiretroviral activities, on the other hand it induces an in vivo transient increase in the inflammation-related remodeling PAF-biosynthetic pathway. This finding supports the hypothesis of inflammation-mediated increased cardiovascular risk in HIV-infected patients during the first months of abacavir-containing HAART.

Vitamin D receptor activators upregulate and rescue podocalyxin expression in high glucose-treated human podocytes.

BACKGROUND: Vitamin D is beneficial in human and experimental chronic kidney disease, the leading cause of which is diabetic nephropathy. Vitamin D through its receptor, VDR, provides renal protection in diabetic nephropathy, but limited data exist about its effect on podocytes. Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. METHODS: We used immortalized human podocytes (human glomerular epithelial cells, HGEC) to assess podocalyxin and nephrin expression after treatment with 1,25-dihydroxyvitamin D3 (calcitriol) and its analogue paricalcitol. The involvement of VDR was investigated by silencing with hVDR-siRNA and ChIP analysis. RESULTS: HGEC exhibit high glucose-mediated downregulation of podocalyxin and nephrin, loss of which has been linked with loss of the permselective renal barrier and proteinuria. Calcitriol and paricalcitol reversed high glucose-induced decrease of nephrin and significantly enhanced podocalyxin expression in podocytes cultured in high glucose. HGEC express VDR and retinoid X receptor (RXR). In the presence of calcitriol and paricalcitol, VDR expression was upregulated and VDR colocalized with RXR in the nucleus. VDR knockdown abolished the protective action of calcitriol and paricalcitol on podocalyxin expression indicating that podocalyxin activation of expression is partly mediated by VDR. Furthermore, VDR specifically regulates podocalyxin expression by bounding to a site upstream of the podocalyxin promoter. CONCLUSION: Vitamin D analogues maintain and, furthermore, re-activate the expression of specialized components of podocytes including podocalyxin, hence they provide protection against loss of the permselective renal barrier, with molecular mechanisms elucidated herein.

Atherosclerosis regression study in rabbits upon olive pomace polar lipid extract administration.

BACKGROUND AND AIMS: Virgin olive oil polar lipid extract (OOPL) and olive pomace polar lipid extract (PPL) have similar antiatherosclerotic effects in cholesterol-fed rabbits. Our aim was to compare the effect of PPL with that of simvastatin on the progression of atherogenesis. METHODS AND RESULTS: Rabbits were fed an atherogenic diet for 6 weeks in order to develop dyslipidemia and atheromatous lesions. Following documentation of these events in random animals (group A, n=6), the remaining were fed for 3 weeks with: standard chow alone (group B, n=6), chow supplemented with PPL (group C, n=6), and chow supplemented with simvastatin (group D, n=6). Blood was collected at 0, 6 and 9 weeks, to determine plasma lipid levels, plasma PAF-AH activity, platelet aggregation (PAF-EC(50)), resistance of plasma to oxidation (RPO) and extent of atheromatous lesions in aortas. The atherogenic diet induced dyslipidemia and increased PAF-AH activity. Dyslipidemia and PAF-activity reduced more effectively in groups C and D. RPO decreased in group B only. PAF-EC(50) values decreased in group C only. Atherogenesis progression in group C was prevented to an extent indistinguishable from that in group D. PAF-AH activity was positively correlated, whereas RPO was negatively correlated with the extent of atheromatous lesions. CONCLUSION: PPL, as a dietary supplement, is equipotent to simvastatin in preventing the progression of atherogenesis.

The implication of platelet activating factor in cancer growth and metastasis: potent beneficial role of PAF-inhibitors and antioxidants.

Cancer is one of the leading causes of death in Europe and United States. New blood vessel formation penetrating into solid tumors seems to be required for their growth and metastasis. Several protein growth factors can induce endothelial cell proliferation and angiogenesis, through signal transduction cascades that result in the production of several inflammatory mediators and lipid second messengers such as prostaglandins and Platelet Activating Factor (PAF). PAF is a potent mediator of inflammation that is implicated in several inflammatory pathological conditions such as atherosclerosis, cardiovascular and renal diseases, allergy, AIDS, cancer etc. It exerts its biological activities through G-protein-coupled receptors. The presence of PAF in the microenvironment of tumors may be due to its synthesis from circulating and / or cancer cells. Moreover, cancer cells and activated endothelial cells expose PAF-receptor on their membrane surface. PAF binding on its receptor induces several pathways that result in the onset and development of tumor induced angiogenesis and metastasis. PAF-receptor antagonists have exhibited promising results in vitro and in vivo as anti-angiogenic molecules in several cancer cells and tumors. A dietary profile reach in antioxidants and PAF-inhibitors (such as the Mediterranean Diet) may provide beneficial preventive and protective effects against development, growth and metastatic manifestations of cancer cells, through either their inhibition of PAF activity and / or its biosynthesis. The clarification of factors that may down regulate pathologically increased PAF-levels in a tumor microenvironment may also contribute to the planning of a potent nontoxic preventive and therapeutic approach against cancer.

Recombinant platelet-activating factor-acetylhydrolase attenuates paracetamol-induced liver oxidative stress, injury, and regeneration.

The aim of this study was to investigate the effects of platelet-activating factor (PAF) inactivator, recombinant PAF-acetylhydrolase (rPAF-AH), on post-paracetamol treatment functional outcome of the liver in the rat. Fifty male Wistar rats were divided into two groups: the control group received a toxic dose of paracetamol (3.5 g/kg body weight [BW]) by gastric tube and the rPAF-AH-treated group received the same dose of paracetamol followed by a dose of rPAF-AH (10 mg/kg BW) intraperitoneally. The animals were sacrificed at time points of 56, 66, 72, 84, and 96 hr after paracetamol treatment. Hepatic injury was evaluated by determination of AST, ALT, and ALP activities and degree of necrosis and apoptosis. Liver regeneration was estimated by [3H]thymidine incorporation into hepatic DNA, liver thymidine kinase activity, and hepatocyte mitotic index. Hepatic levels of malondialdehyde (MDA) and serum cholesterol/high-density lipoprotein cholesterol fraction were also measured as parameters of oxidant-antioxidant balance. The positive effects of rPAF-AH were expressed by (1) reduction of oxidative stress, (2) large decrease in hepatic injury, and (3) diminution of regenerating activity. These results indicate that the use of PAF inactivator enhances the liver's recovery from paracetamol intoxication and attenuates the severity of experimental liver injury, providing important means of improving liver function following paracetamol intoxication.

Effect of platelet-activating factor (PAF) receptor antagonist (BN52021) on acetaminophen-induced acute liver injury and regeneration in rats.

BACKGROUND: Platelet-activating factor (PAF) is an endogenous lipid mediator that plays a key role in catalyzing various pro-inflammatory processes associated with acute liver injury. In the present study, the possible influence of PAF-R antagonist (BN52021) on the protection of liver injury after 4-hydroxyacetanilide, N-acetyl-p-aminophenol, paracetamol (APAP) intoxication was investigated. METHODS: Thereby, one group of rats was treated with a toxic dose of APAP (3.5 g/kg body weight (b.w.). The animals were killed at 56, 66, 72, 84 and 96 h after treatment. RESULTS: APAP was found to cause an acute hepatic injury, evident by alterations of biochemical (serum enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase) and liver histopathological (degree of necrosis and apoptosis) indices, which was followed by liver regeneration, evident by three independent indices ([3H] thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index). The protective effects of BN52021 were qualified during post-treatment time by: (1) significant reduction of hepatic injury as showed by all biochemical and histological parameters, (2) high decrease of regenerating activity showed by three regenerative markers and (3) remarkable increase of PAF-acetylhydrolase (PAF-AH) activity. CONCLUSION: These results suggest that PAF may play an important role in APAP-induced liver injury and regeneration, and PAF-R antagonist (BN52021) attenuates liver damage.

Platelet-activating factor (PAF) involvement in acetaminophen-induced liver toxicity and regeneration.

Acetaminophen-induced toxicity has been attributed to cytochrome P-450-generated metabolites, which covalently modify target proteins. However, the mechanism of liver injury pathogenesis needs to be further elucidated. Platelet-activating factor (PAF) is one of the mediators involved in inflammatory tissue alterations associated with acute liver failure. In this study, alterations in blood PAF levels and the serum activity of PAF-acetylhydrolase (PAF-AH) were investigated over the time course of liver injury and regeneration induced by acetaminophen treatment in rats. The administration of a toxic dose of acetaminophen (3.5 g/kg) in rats caused acute hepatic injury, as evident by alterations of biochemical (serum enzymes: ALT, AST and ALP) and liver histopathological (degree of inflammation and apoptosis) indices between 20 and 40 h post-treatment. The hepatic damage was followed by liver regeneration, made evident by three independent indices ([3H]thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index), presenting a peak at 72 h. The PAF levels were elevated at 24 and 28 h, presenting a remarkable peak at 32 h post-treatment. PAF-AH activity presented different kinetics to that of PAF. The enzyme activity was relatively low at all time points examined before the rise in PAF activity, peaking later, at 72, 84 and 96 h. Our data demonstrate that PAF is involved in the pathogenesis of acute liver failure and in augmented compensatory liver tissue repair post-acetaminophen treatment. However, the putative role of PAF during liver toxicity and regeneration remains to be established.

Biological activity of total lipids from red and white wine/must.

Wine is an essential component of the Mediterranean diet, and it is thought to exert a protective effect against coronary heart disease. Although many efforts have been made to determine the protective compounds in wines, their exact nature and how they are involved in the protection mechanisms are still unclear. In this study, total lipids, total polar lipids, and total neutral lipids of five wines and three musts were tested in vitro for their ability to induce washed rabbit platelet aggregation and/or to inhibit platelet activating factor (PAF) induced aggregation. The results showed that the biological activity of wine/must total lipids can be attributed mainly to total polar lipids. In the red wine Cabernet Sauvignon, we fractionated total neutral lipids, total polar lipids, and pigments by HPLC. Each fraction was tested in vitro for its biological activity. Structural data of the most active fractions, based on biological, chemical, and spectral methods, are also presented.

Separation of biologically active lipids from red wine.

A number of lines of evidence suggest that red wine exerts a protective effect against coronary heart disease, but the nature of the protective compounds is unclear and the mechanism is incompletely understood. In this study, total lipids of a Greek red wine were separated into neutral and polar lipids. Polar lipids were further separated into glyco- and phospholipids, which were fractionated by HPLC. Each lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF) and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. A significant number of glyco- and phospholipids that exerted the above biological activities were detected. Structural data of an active phosphoglycolipid are also provided. trans-Resveratrol demonstrated also a dose-dependent inhibition of PAF-induced platelet aggregation along with the already reported inhibitory activity against thrombin and adenosine-5'-diphosphate. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in red wine may contribute to the protective role of red wine against atherosclerosis.

Lipid fractions with aggregatory and antiaggregatory activity toward platelets in fresh and fried cod (Gadus morhua): correlation with platelet-activating factor and atherogenesis.

Cod (Gadus morhua) is a popular part of the diet in many countries on both sides of the North Atlantic; in most cases it is consumed fried. In this study, total lipids of cod muscle were separated into neutral and polar lipids, which were further fractionated by HPLC. The lipid fractions were tested in vitro, against washed rabbit platelets, for the probable existence of lipid compounds that either exhibit an action similar to that of platelet-activating factor (PAF) or inhibit the action of PAF. The platelet bioassay was used to evaluate total lipids, total polar lipids, and total neutral lipids, before any further separation. Detection of these compounds in fresh and fried cod could be used to evaluate the nutritional value of this important fish. The in vitro biological study of lipids showed that in fresh cod lipid fractions, ranges of PAF-like and anti-PAF-like activities were present, whereas in fried cod lipid fractions, both neutral and polar, anti-PAF activities were mainly observed. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in cod may contribute to the possible protective role of fish, in this case cod, against atherosclerosis.

Identification of a new endogenous platelet-activating factor-like molecule in gingival crevicular fluid.

Periodontal disease is an inflammatory disease and the major cause of tooth loss in adults. Bacteria and their products are the causative agents of this disease. Endogenous molecules mediate the inflammatory process and play a major role in its amplification and perpetuation as well as in the ensuing tissue destruction. The relationship between platelet-activating factor (PAF) and periodontal disease has not so far been examined thoroughly. We have isolated a phospholipid molecule with PAF-like activity from gingival crevicular fluid. This molecule, purified on HPLC, causes washed platelet aggregation with EC50 value 0.1 microM, based on phosphorus determination. It acts through PAF-receptors and is inactivated by PAF-acetylhydrolase. In addition, this phospholipid presents biological activity towards human platelets. The combination of the results obtained from the chemical and enzymic treatments, the biological assays as well as results from the electrospray analysis, leads to the conclusion that this phospholipid is a hydroxyl-PAF analogue with relative molecular mass 703. This PAF-like molecule may be implicated in periodontal disease.

Synthesis of a new phosphoglycolipid with biological activity towards platelets.

Various synthetic as well as naturally occurring compounds have been found to exhibit platelet-activating factor (PAF)-like activity or to act as specific PAF inhibitors. In this work we have synthesized a new phosphoglycolipid, methyl 2,3,4-tri-O-acetyl-6-(1'-O-stearoyl-2'-O- acetyl-DL-glycero-3'-phosphoryl)-alpha-D-glucopyranoside ammonium salt, using a combination of known synthetic steps. This phosphoglycolipid was first purified on TLC (Rf 0.7, using chloroform/methanol/water, 65:25:4, v/v/v as solvent system). It was further purified onto a high performance liquid chromatography silica column with an elution system that contained acetonitrile and methanol (retention time 13.5 min). Its identification was based on chemical determinations and electrospray mass spectrometry analysis. The above compound induced washed platelet aggregation with an EC50 value at 2 x 10(-4) M. The aggregation curve was biphasic, the first wave of which was through the PAF way while the second one was through the ADP way. Treatment with acetylhydrolase resulted in a rapid decrease of the first wave of aggregation and in a slow decrease of the second wave. In lower concentrations, the phosphoglycolipid inhibited PAF- and thrombin-induced aggregation with IC50 values of the order of 10(-7) M. In conclusion, this phosphoglycolipid has a diverse biological activity. The PAF-like activity of this new lipid enforces the conception that PAF is a member of a large family consisting of lipid mediators.

Blood cardiolipin in haemodialysis patients. Its implication in the biological action of platelet-activating factor.

Bovine heart cardiolipin specifically inhibits platelet aggregation induced by PAF in vitro. In the past we have reported that patients with primary glomerulonephritis have increased PAF levels in plasma (Iatrou et al., 1995b). In this work we investigate the existence of cardiolipin in the blood of end-stage renal patients due to primary glomerulonephritis and we study its implication in the biological study of PAF. Lipids from blood samples of end-stage renal patients were extracted, fractionated onto silicic acid column and onto High Performance Liquid Chromatography (HPLC) cation exchange column. PAF fraction was removed and phospholipids were separated from the rest lipid fraction with current counter distribution and furthermore fractionated onto HPLC silica column. The results show: 1. cardiolipin is present in the blood of end-stage renal patients. 2. Blood cardiolipin specifically inhibits PAF-induced aggregation in washed rabbit platelets. 3. Scatchard plot analysis of PAF binding, in the presence of unlabelled PAF and in the presence of cardiolipin, shows that rabbit platelets possess two different types of binding sites. One of which is saturable and of high affinity, kD = 0.103 +/- 0.03 nM (SEM, n = 3) with 337 +/- 94 binding sites per platelet for PAF and kD = 0.087 +/- 0.02 nM with 371 +/- 92.7 binding sites per platelet for cardiolipin while the other one has almost infinite binding capacity. 4. Blood cardiolipin competes [3H]PAF binding in rabbit platelets. This work shows that cardiolipin exists in the blood of end-stage renal patients and specifically inhibits PAF-induced aggregation as well as PAF binding in rabbit platelets. The possible implication of the biological actions of cardiolipin in the anticardiolipin-antiphospholipid syndrome is also discussed.

The biological activity of acetylated sphingosylphosphorylcholine derivatives.

Taking into account their stereochemical similarity with 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine (PAF), which is known to exhibit a diverse spectrum of biological actions, including platelet aggregating and glycogenolytic activity, the acetylated derivatives of sphingosylphosphorylcholine and sphingomyelin were synthesized and tested for their ability to promote washed rabbit platelet aggregation and glycogenolysis in Tetrahymena pyriformis cells. Sphingomyelin (SPM) and sphingosylphosphorylcholine (SPC) were subjected to acetylation, following chromatographic purification and physicochemical characterization. The synthesized compounds N-acetyl, O-acetyl SPC (NAc, OAc SPC), N-acetyl SPC (NAc SPC) and O-acetyl SPM (OAc, SPM) were tested for their biological activity in the washed rabbit platelets and washed Tetrahymena pyriformis cell systems. These derivatives induced [3H]serotonin and ATP release and a monophasic aggregation of washed rabbit platelets in the concentration range 10(-8)-10(-6) M. However, authentic PAF-induced washed rabbit platelet aggregation was inhibited at higher concentrations of the acetylated sphingophospholipid compounds ( > 10(-6) M) and by the PAF-specific receptor(s) antagonists kadsurenone and triazolam. Platelet desensitization and crossed desensitization experiments with authentic PAF and the acetylated sphingophospholipids, suggested interaction with the same receptor(s) as PAF and different from the ADP or thrombin receptor. The involvement of calmodulin and protein kinase C in the biological activity of the prepared analogues was also demonstrated. Besides their action on rabbit platelets, the PAF-like activity of the acetylated sphingophospholipids was also demonstrated in a platelet-independent system, by showing that they stimulate glycogenolysis in washed Tetrahymena pyriformis cells. These observations indicate that a new class of compounds with PAF-like activity were synthesized but their role in the cellular metabolism remains to be shown. The existence of acetylated sphingophospholipids with PAF-like activity has so far been reported only in Urtica dioica.

Platelet-activating factor levels and PAF acetylhydrolase activities in patients with primary glomerulonephritis.

Platelet-activating factor (PAF) belongs to a new class of lipid chemical inflammatory mediators described as acetyl glyceryl ether phosphorylcholine. Although PAF has been implicated in kidney injury, its role in renal immune injury has not been clearly defined yet. We studied the levels of PAF in the plasma and urine and acetylhydrolase (AH) activities in serum and in renal tissue (cortex, C and medulla, M) in patients with primary glomerulonephritis (PGN). PAF levels in the plasma and urine and AH activity in serum of normal volunteers as well as AH activities in normal renal parenchyma (C and M) from nephrectomized patients (served as control) were also measured. Our results demonstrate increased PAF levels in the plasma and urine as well as increased AH activity in serum in patients with PGN in comparison to normal volunteers. AH activity in cortex of those patients was diminished compared to normal kidney tissue. We propose that the enhanced AH activity in serum in patients with PGN could be due to hyperproduction and low degradation of PAF in nephritic tissue which on one hand results in enhanced PAF levels in the plasma and in urine and on the other hand results in enhanced serum AH activity by virtue of substrate (PAF) availability. These data provide new knowledge in the homeostasis of PAF and its degrative enzyme in the setting of PGN.

The production of platelet-activating factor (PAF) during hemodialysis with cuprophane membrane. Does the calcium concentration in the dialysate play any role on it?

There is evidence that PAF may be produced during hemodialysis (HD) mainly when using cuprophane membrane (CU). It is also known that PAF production is dependent on the amount of extracellular calcium (ECa2+). In the present study, we investigated the production of PAF during HD with CU as well as the role of the Ca2+ in the dialysate with respect to PAF production. Five hemodialyzed patients were studied in two consecutive HD sessions (the first performed using dialysate without Ca2+ and the second with a Ca2+ concentration of 3.25 mEq/L) and at different times during the sessions the circulating PAF levels as well as the leukocyte and platelet counts were measured. The results demonstrated that a) PAF was indeed produced during HD with CU, b) the highest PAF levels in blood were observed between 5 and 15 minutes from the beginning of HD, at which time the lowest circulating leukocyte and platelet count were measured and c) PAF levels in blood were inversely proportional to the Ca2+ concentration in the dialysate (with the exceptional case of the 15 minutes), although we expected the opposite results.