Alexandros B. Tsoupras

Characterization of the De Novo Biosynthetic Enzyme of Platelet Activating Factor, DDT-Insensitive Cholinephosphotransferase, of Human Mesangial Cells

Platelet activating factor (PAF), a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT) of human mesangial cell (HMC), the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA), dithiothreitol (DTT), divalent cations (Mg2+ and Ca2+), EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid) and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass “Dicentrarchus labrax,” and gilthead sea bream “Sparus aurata”). The results indicated that the above compounds can influence PAF-CPT activity of HMC.

Anti-Platelet-Activating Factor Effects of Highly Active Antiretroviral Therapy (HAART): A New Insight in the Drug Therapy of HIV Infection?

Platelet-activating factor (PAF) is a potent inflammatory mediator, which seems to play a role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposis sarcoma, and HIV-related nephropathy. PAF antagonists have been studied in these conditions with promising results. In order to examine the possible interactions between PAF and antiretroviral therapy, we studied the effect of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors against PAF biological activities and its basic biosynthetic enzymes dithiothreitol-insensitive PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), as well as its main degradative enzyme PAF-acetylhydrolase, of human mesangial cell line (HMC). We also studied the effect of several backbones and highly active antiretroviral therapy (HAART) regimens against PAF activity. Among the drugs tested, several inhibited PAF-induced platelet aggregation in a concentration-depended manner, with tenofovir, efavirenz, and ritonavir exhibiting the higher inhibitory effect. In addition, when these drugs were combined in backbones and HAART regimens based on American antiretroviral therapy proposals, they also synergistically exhibited an inhibitory effect against PAF-induced platelet aggregation. Several of these drugs have also inhibited in vitro microsomal PAF-CPT activity, and concentrations of lopinavir-r or tenofovir-DF (similar to their IC(50) against PAF-induced platelet aggregation) exhibited the same effect against PAF-CPT and Lyso-PAF-AT when added in the cell medium of cultured HMC. In addition, in naïve patients treated with one of the most potent anti-PAF HAART regimens (efavirenz/emtricitabine/tenofovir-DF) for a period of 1 month, a significant reduction of the specific activity of PAF-CPT of washed human leukocytes of these patients was also observed, compared with its levels before the HAART treatment. These promising results need to be further studied and confirmed by additional in vivo tests in order to optimize HAART efficacy.

In Vitro and In Vivo Effects of Statins on Platelet-Activating Factor and Its Metabolism

Platelet activating factor (PAF) is implicated in cardiovascular disease (CVD). Statins are widely used in these situations. Therefore, we assessed their effect on the biological activities and metabolism of PAF. Several statins, including simvastatin, exhibited an inhibitory effect against PAF, comparable with that of PAF-inhibitors. Simvastatin also suppressed in vivo PAF-biosynthesis via the de novo pathway, in leukocytes of 6 simvastatin-treated volunteers. Total cholesterol and low-density lipoprotein cholesterol were also significantly decreased, whereas high-density lipoprotein cholesterol, triacylglycerol, EC50, and lag time were unaffected in these participants. Simvastatin with an intact lactone ring also inhibited PAF-activities, while incubation of human mesangial cells with it also resulted in decreased de novo PAF-biosynthesis. This suggests that these simvastatin-dependent effects are independent of its lactone ring. These new actions of statins should be further studied in PAF-implicated pathological conditions such as CVD, cancer, and renal disease.

Platelet-Activating Factor and Its Basic Metabolic Enzymes in Blood of Naive HIV-Infected Patients

Platelet-activating factor (PAF), a mediator of proatherosclerotic inflammatory processes, is also implicated in endothelial dysfunction during human immunodeficiency virus (HIV) infection. We examined PAF metabolism in blood of naive male patients, 8 with early HIV infection (group A) and 17 just before treatment initiation (group B), versus 18 healthy age-matched males (group C). Statistical analysis was performed with 1-way analysis of variance (ANOVA) criterion and Pearson r test. Higher PAF biosynthesis in patients’ leukocytes versus group C was accompanied by an increase in lipoprotein-associated phospholipase A2 (Lp-PLA2) activity that degrades PAF. Moreover, PAF synthesis was higher and Lp-PLA2 activity was lower in group B compared to group A. Lipoprotein-associated phospholipase A2 was positively correlated with viral load and negatively correlated with CD4 cell counts in group B. The activities of PAF-basic biosynthetic enzymes in patients’ leukocytes were also negatively correlated with CD4 cell counts. The observed continuous increase in PAF biosynthesis during HIV infection progress seems to amplify the risk of AIDS manifestations and/or cardiovascular complications in HIV-infected patients, while a subsequent increase in Lp-PLA2 activity seems to be a host response.

Paricalcitol effects on activities and metabolism of Platelet Activating Factor and on inflammatory cytokines in hemodialysis patients

Purpose Paricalcitol improves the inflammatory status of hemodialysis patients. PAF is a strong inflammatory mediator which is produced during hemodialysis. We studied the effects of paricalcitol on PAF and other inflammatory mediators implicated in chronic kidney disease (CKD). Methods We examined the in vitro effects of paricalcitol on PAF/thrombin-induced aggregation as well as on the activities of PAF-basic metabolic enzymes, lyso-PAF acetyltransferase (Lyso-PAF-AT), DTT-insensitive CDP-choline: 1-alkyl-2-acetyl-sn-glycerol cholinephospho-transferase (PAF-CPT) and PAF-acetylhydrolase (PAF-AH) in blood cells from healthy volunteers. In addition, the in vivo effects of paricalcitol on the above these enzymes were examined in plasma and blood cells of hemodialysis patients who had not received any type of vitamin D treatment during the last three months before and after receiving paricalcitol for a month. Finally, IL-12p70, IL-1β, IL-6, IL-8 and TNF-α were measured. Results Paricalcitol inhibited in vitro PAF/thrombin-induced platelet aggregation and the inhibitory effect was comparable with that of PAF/thrombin antagonists. In addition, paricalcitol inhibited in vitro PAF-CPT activity in platelets and leukocytes and increased PAF-AH activity in leukocytes, while much higher concentrations of paricalcitol were needed to inhibit Lyso-PAF-AT activity. Similarly, in hemodialysis patients, paricalcitol treatment reduced PAF-CPT activity in platelets and leukocytes and increased PAF-AH activity in leukocytes, while it could not influence Lyso-PAF-AT activity. On the other hand, paricalcitol therapy reduced IL-8, IL-1β, and TNF-α. Conclusions These results further support the beneficial effects of vitamin D treatment in hemodialysis patients, since it strongly affects PAF/thrombin activities, PAF-metabolism, and IL-8, IL-1β and TNF-α circulating levels.

Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet-activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In this study, hydroxyl-PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of hydroxyl-PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.

Dairy Fats and Cardiovascular Disease: Do We Really Need to Be Concerned?

Cardiovascular diseases (CVD) remain a major cause of death and morbidity globally and diet plays a crucial role in the disease prevention and pathology. The negative perception of dairy fats stems from the effort to reduce dietary saturated fatty acid (SFA) intake due to their association with increased cholesterol levels upon consumption and the increased risk of CVD development. Institutions that set dietary guidelines have approached dairy products with negative bias and used poor scientific data in the past. As a result, the consumption of dairy products was considered detrimental to our cardiovascular health. In western societies, dietary trends indicate that generally there is a reduction of full-fat dairy product consumption and increased low-fat dairy consumption. However, recent research and meta-analyses have demonstrated the benefits of full-fat dairy consumption, based on higher bioavailability of high-value nutrients and anti-inflammatory properties. In this review, the relationship between dairy consumption, cardiometabolic risk factors and the incidence of cardiovascular diseases are discussed. Functional dairy foods and the health implications of dairy alternatives are also considered. In general, evidence suggests that milk has a neutral effect on cardiovascular outcomes but fermented dairy products, such as yoghurt, kefir and cheese may have a positive or neutral effect. Particular focus is placed on the effects of the lipid content on cardiovascular health.

Effects of Highly Active Antiretroviral Therapy on Platelet Activating Factor Metabolism in Naïve 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.

In vivo effect of two first-line ART regimens on inflammatory mediators in male HIV patients

BackgroundPersistent immune activation and inflammation are lying behind HIV-infection even in the setting of ART mediated viral suppression. The purpose of this study is to define the in vivo effect of two first-line ART regimens on certain inflammatory mediators in male HIV patients.MethodsMale, naive, HIV-infected volunteers were assigned either to tenofovir-DF/emtricitabine/efavirenz (Group_T) or abacavir/lamivudine/efavirenz (Group_A). Platelet Activating Factor (PAF) levels and metabolic enzymes together with HIV-implicated cytokines (IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNFa) and VEGF were determined for a 12-month period. Differences within each group were determined by non-parametric Friedman and Wilcoxon test, while the differences between the groups were checked by ANOVA repeated measures.ResultsBoth ART regimens present pronounced effect on inflammatory mediators, resulting in decreased PAF levels and Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity for tenofovir-containing regimen and same as baseline PAF levels with a peak though at the 3rd month as well as elevated Lp-PLA2 activity for abacavir-containing regimen.ConclusionsStudies regarding the effect of first-line ART regimens on inflammation may be beneficial in preventing chronic morbidities during HIV-treatment. From this point of view, the present study suggests an anti-inflammatory effect of tenofovir-containing ART, while the temporary increase of PAF levels in abacavir-containing ART may be the link between the reported cardiovascular risk and abacavir administration.

Inflammation, not Cholesterol, Is a Cause of Chronic Disease

Since the Seven Countries Study, dietary cholesterol and the levels of serum cholesterol in relation to the development of chronic diseases have been somewhat demonised. However, the principles of the Mediterranean diet and relevant data linked to the examples of people living in the five blue zones demonstrate that the key to longevity and the prevention of chronic disease development is not the reduction of dietary or serum cholesterol but the control of systemic inflammation. In this review, we present all the relevant data that supports the view that it is inflammation induced by several factors, such as platelet-activating factor (PAF), that leads to the onset of cardiovascular diseases (CVD) rather than serum cholesterol. The key to reducing the incidence of CVD is to control the activities of PAF and other inflammatory mediators via diet, exercise, and healthy lifestyle choices. The relevant studies and data supporting these views are discussed in this review.