Effects of HAART on Platelet-Activating Factor Metabolism in Naive HIV-Infected Patients I: Study of the Tenofovir-DF/Emtricitabine/Efavirenz HAART Regimen

Abstract

Platelet-activating factor (PAF) is implicated in human immunodeficiency virus (HIV)-related manifestations. Increased PAF synthesis has been recently detected in HIV-infected patients. In this study, we examined in naive HIV-infected patients the in vivo effects of a highly active antiretroviral therapy (HAART) regimen, containing tenofovir-DF/emtricitabine/efavirenz, on PAF metabolism. The specific activities of PAF basic biosynthetic enzymes, PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (lyso-PAF-AT), but also the ones 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 eight asymptomatic naive 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 measures ANOVA and the Pearson r criterion were used to study statistical differences and correlations–partial correlations, while linear mixed models were conducted in order to estimate association(s) between time-dependent changes in these factors. Before treatment, the activities of PAF-CPT in leukocytes and LpPLA₂ in plasma were found to be inversely correlated with CD4 cell counts and positively correlated with the viral load. After 6 months of treatment, the activities of basic PAF-biosynthetic enzymes, PAF-CPT and lyso-PAF-AT, were both reduced in leukocytes. At 6 months, PAF-AH activity was also reduced in these cells, while LpPLA₂ remained stable. The reduction of PAF-CPT occurred even from the first month, while there is a time-dependent correlation between the increase of CD4 and the decrease of both viral load and PAF-CPT of leukocytes during treatment. Apart from its classical antiretroviral activities the tenofovir-DF/emtricitabine/efavirenz regimen also exhibited favorable effects on PAF metabolism and therefore may also display beneficial effects in some HIV-related conditions, such as cardiovascular disease (CVD), in which PAF is implicated.

Introduction

P latelet-activating factor (PAF) is a phospholipid signaling molecule of the immune system and a significant mediator of inflammation.^1,2 PAF acts through specific membrane receptors coupled with G-proteins (PAF-receptor)² and transmits outside-in signals to intracellular transduction systems in a variety of cell types, including key cells of the innate immune and hemostatic systems: neutrophils, monocytes, and platelets.³

Normally, PAF levels in cells, tissues, and blood of higher organisms are regulated by a group of PAF metabolic enzymes. Two different and distinct enzymatic routes of PAF biosynthesis have been reported, namely the de novo pathway and the remodeling one.^4
–6 In the de novo pathway, a central step is the conversion of 1-O-alkyl-2-acetylglycerol to PAF by a specific dithiothreitol-insensitive CDP-choline: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT, EC 2.7.8.16). In the remodeling pathway, first a cytoplasmic phospholipase A₂ yields lyso-PAF from 1-O-ether-linked membrane phospholipids, which in turn is then acetylated by a lyso-PAF:acetyl-CoA acetyltransferase (lyso-PAF-AT, EC 2.3.1.67) in order to produce PAF. The main enzyme involved in the catabolism of PAF is a PAF-specific acetylhydrolase (PAF-AH, EC 3.1.1.47), also known in plasma as lLipoprotein-associated phospholipase A₂ (Lp-PLA₂), which cleaves the short acyl chain at the sn-2 position and forms the biologically inactive lyso-PAF.⁷

Dysregulation of PAF metabolism induces altered PAF levels that may lead to inflammatory manifestations. Therefore, PAF seems to be implicated in several pathological conditions such as cardiovascular,^8,9 renal,¹⁰ and periodontal diseases,¹¹ allergy,¹² diabetes,¹³ cancer,¹⁴ and AIDS.^15

–19 Several PAF inhibitors have been studied in these conditions with promising results.^20,21

In HIV infection PAF seems to play a significant role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposi's sarcoma, and HIV-related nephropathy.^15
–17 In HIV-related manifestations altered host cells and their secreted subproducts such as Tat protein induce PAF synthesis via the effect of cytokines (such as TNF-α) and growth factors (such as VEGF), while several PAF antagonists have been found to prevent these effects.^15

–18 Thus PAF seems to be implicated in monocytic cell recruitment and in increased vascular permeability, two major biological events that take place in the initiation of various HIV-related manifestations.

We have previously described the in vivo altered metabolism of PAF during HIV infection in naive patients.²² In asymptomatic patients just before initiation of highly active antiretroviral therapy (HAART) but also in patients with early HIV infection, the activities of the key enzymes of PAF biosynthetic pathways in their leukocytes, PAF-CPT of the de novo and lyso-PAF-AT of the remodeling pathways, were found to be higher than those of control subjects. The activation of PAF formation in these patients was found to be followed by an increase in the activity of LpPLA₂, probably as a response to elevated PAF levels.²² In addition, we have also indicated that several antiretroviral drugs and HAART regimens, apart from their general antiretroviral activity, exhibited an inhibitory effect against PAF biological activities in washed rabbit platelets and also inhibited PAF-CPT activity of cultured human mesangial cells. Antiretroviral drugs and HAART regimens with the most potent anti-PAF activity inhibited also both PAF-CPT and lyso-PAF-AT activities when added to the cell medium of these cells.¹⁹ Moreover, preliminary in vivo results in three naive patients treated for 1 month with the HAART regimen tenofovir-DF/emtricitabine/efavirenz revealed a significant reduction of PAF-CPT activity in leukocytes.¹⁹

In the present study we have further investigated in HIV-infected patients the in vivo effect of a HAART regimen containing tenofovir-DF/emtricitabine/efavirenz on PAF metabolism. For this purpose we have measured the specific activities of PAF key metabolic enzymes, PAF-CPT, lyso-PAF-AT, and PAF-AH, in leukocytes and platelets as well as LpPLA₂ in plasma of eight naive male HIV-infected patients before and after the initiation of HAART for a period of 6 months. We have also studied any potential correlation between these enzyme activities and other biochemical parameters such as viral load, CD4⁺ cell counts, lipid profile, and hepatic enzymes.

Materials and Methods

Materials and instruments

Centrifugations were performed in an Heraeus Labofug 3L-R, an Heraeus Labofug 400R (Heraeus Hanau, Germany), and a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont de Nemours & Co., Inc., Sorvall Instruments Division, Newtown, CT) and a refrigerated Micro 22R Zentrifugen Hettich superspeed centrifuge (Hettich, Kirchlengern, East Westphalia, Germany). Homogenizations were conducted in 30% of power of a supersonic Bandelin Sonoplus HD 2070 sonicator (BHD, Berlin, Germany). The liquid scintillation counter used was a 1209 Rackbeta (Pharmacia, Wallac, Finland). Platelet aggregation studies were performed in a model 400 VS aggregometer of Chrono-Log (Chrono-Log, Havertown, PA) coupled to a Chrono-Log recorder at 37°C with constant stirring at 1200 rpm.

Bovine serum albumin (BSA), PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine), trichloroacetic acid (TCA), CDP-choline, lyso-PAF, acetyl-CoA, dithiothreitol (DTT), EDTA, MgCl₂, Tris, and analytical reagents and solvents were purchased from Sigma (Sigma, St. Louis, MO). 1-O-Hexadecyl-2-[³H]acetyl-sn-glycerol-3-phosphocholine ([³H]PAF) with a specific activity of 10 Ci/mmol was obtained from New England Nuclear (NEN, Dupont, Boston, MA). 1-O-alkyl-2-sn-acetylglycerol (AAG) was purchased from BIOMOL International LP (BIOMOL-ILP, Palatine House, Matford Court, Exeter, UK). 2,5-Diphenyloxazole (PPO) and 1,4-bis(5-phenyl-2-oxazolyl) benzene (POPOP) were purchased from BDH Chemicals (BDH, Dorset, England). Scintillation liquid cocktail (dioxane base) was prepared by diluting 7 g PPO, 0.3 g POPOP, and 100 g naphthalene in 200 ml H₂O and then transferred to 1 liter of dioxane. Liquid chromatography grade solvents and silica G for TLC were purchased from Merck KGaA (Merck, Darmstadt, Germany).

CD4 cell counts were automatically calculated using the Tetra One System on the Beckman Coulter EPICS XL flow cytometer (Beckman Coulter, Nyon, Switzerland), while viral load was determined using the Versant HIV-1 RNA 3.0 assay (bDNA). The kit for this assay was purchased by Siemens Healthcare Diagnostics (Siemens, Tarrytown, NY).

Serum total cholesterol (TC), HDL-cholesterol, LDL-cholesterol, triglycerides, oxaloacetic transaminase (AST), pyruvic transaminase (ALT), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT), creatinine, and all the other biochemical parameters were measured in a Siemens Dimension RxL automatic analyzer (Siemens Center, 60 MacPherson Road, Singapore).

Study design

Study enrollment began after obtaining approval from the local ethics committee. All patients signed the informed consent of participation in the study. They were randomly recruited from the 3rd Internal Medicine Department–Infectious Diseases Unit, Red Cross General Hospital, Athens, Greece. All subjects were male (n=8) and had evidence of HIV infection as determined by the presence of antibodies against HIV measured by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. AIDS was identified on the basis of the revised criteria of the Centers for Disease Control. These patients were asymptomatic and fulfilled the criteria for HAART initiation, based on their levels in both CD4 cell counts and viral load. These criteria were applied according to European (European AIDS Clinical Society Guidelines, version 5, 2009) and international guidelines (Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents, Department of Health and Human Services. December 1, 2009; 1–161. Available at http://www.aidsinfo.nih.gov/ContentFiles/Adultand AdolescentGL.pdf).

Subjects with symptoms or signs of acute infection, or other inflammatory manifestations such as periodontal or renal diseases, were excluded. None of the subjects had diabetes, renal failure, nephritic syndrome, active hepatitis, or cirrhosis, or took medication for treatment of hyperlipidemia at the time of the study.

Patients received the HAART regimen tenofovir-DF/emtricitabine/efavirenz and blood samples were collected before (baseline defined as 0 months) and after 1, 3, and 6 months of treatment initiation.

Isolation of plasma, leukocytes, and platelets from blood samples

Blood was obtained from the patients after a 10-h overnight fast. Plasma, leukocytes, and platelets were isolated as previously described¹⁹ with some modifications. Briefly: An amount of 9 ml blood was obtained from each volunteer in 1 ml of an anticoagulant solution of sodium citrate/citrate acid. The sample was centrifuged at 194×g for 10 min at 25°C (first centrifugation). Then three simultaneous procedures were carried out.

Isolation of plasma

The supernatant of the first centrifugation (plasma rich in platelets) was centrifuged at 1400×g for 20 min at 25°C (second centrifugation) in order to obtain plasma. Plasma was further centrifuged at 20,000×g for 1 h at 4°C in order to remove the viral load, was aliquoted, and stored at –80°C until the time of the LpPLA₂ assay analysis.

Isolation of human platelets (HPs)

The pellet of the second centrifugation (platelets) was washed once with saline and after centrifugation at 1400×g for 20 min at 25°C was resuspended in 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.4). Subsequently, platelets were sonicated on ice (4×15 s) and centrifuged at 500×g for 10 min at 4°C. Platelet homogenates after protein determination by the Bradford method²³ were aliquoted and stored at –80°C.

Isolation of human leukocytes (HLs)

In the pellet of the first centrifugation (leukocytes and erythrocytes) saline was added until the volume of 10 ml and, after mixing by inversion, the solution was separated in two parts of 5 ml each. In each part the isolation of the leukocytes from the contaminating erythrocytes was achieved by erythrocyte sedimentation. An aliquot of 1.7 ml of dextran solution (3% dextran in NaCl 0.15 M) was added in each part and the mixtures were kept for 1 h at room temperature. The leukocyte-rich supernatants of the two aliquots were pooled and centrifuged at 500×g for 10 min at 25°C. The supernatant was removed and contaminating erythrocytes of the sediment pellet were lysed with the addition of 5 ml of a lysis solution consisting of 155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA and then removed by centrifugation at 300×g for 10 min at 25°C. The isolated leukocytes were resuspended in 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.4), sonicated on ice (4×15 s), and centrifuged at 500×g for 10 min at 4°C. Leukocyte homogenates after protein determination by the Bradford method²³ were aliquoted and stored at –80°C.

Assays

PAF-CPT activity assays

The assay was performed in leukocyte and platelet homogenates as previously described.²⁴ Briefly, the reaction was carried out at 37°C for 20 min in a final volume of 200 μl containing 100 mM Tris-HCl (pH 8.0), 15 mM dithiothreitol (DTT), 0.5 mM EDTA, 20 mM MgCl₂, 1 mg/ml BSA, 100 μM CDP-choline, 100 μM 1-O-alkyl-2-sn-acetyl-glycerol (AAG, added in the assay mixture in ethanol), and the sample (0.05 mg/ml final concentration of protein). The mixture of Tris, DTT, EDTA, MgCl₂, and BSA was incubated in 37°C for 5 min. Initially, the homogenate was added in the mixture. After 30 s, AAG was added and 30 s later the reaction was started by the addition of CDP-choline. The reaction was stopped by adding 0.5 ml of cold methanol (2% in acetic acid) after 20 min.

Lyso-PAF-AT activity assays

The assay was performed in leukocyte and platelet homogenates as previously described.¹⁹ Briefly, the reaction was carried out at 37°C for 30 min in a final volume of 200 μl containing 50 mM Tris-HCl (pH 7.4), 0.25 mg/ml BSA, 20 μM lyso-PAF, and 200 μM acetyl-CoA and the sample (0.125 mg/ml final concentration of protein). The reaction was started by the addition of the homogenated sample and was stopped after 30 min by adding 0.5 ml of cold methanol (2% in acetic acid).

Determination of produced PAF and biosynthetic enzymes (PAF-CPT and lyso-PAF-AT) activities

The extraction, purification, and determination of the produced PAF in each assay were carried out as previously described.^19,24 Briefly, PAF was extracted according to the Bligh–Dyer method²⁵ and was separated by thin-layer chromatography (TLC) on Silica Gel G-coated plates with a development system consisting of chloroform:methanol:acetic acid:water (100:57:16:8, v/v/v/v). The PAF band was scrapped off and extracted²⁴ and the amount of PAF was determined by the washed rabbit platelet aggregation assay.¹

All assays were performed in duplicate and specific activities of PAF-CPT and lyso-PAF-AT were expressed as pmol of produced PAF/min/mg of sample protein present in each assay.

PAF-acetylhydrolase activity assays

PAF-AH in HLs and HPs as well as Lp-PLA₂ in plasma were determined by the TCA precipitation method using [³H]PAF as a substrate, as previously described.²⁶ Briefly, 0.05 mg of protein from HLs homogenates or 0.1 mg of protein from HPs homogenates or 2 μl of plasma was incubated with 4 nmol of [³H]PAF (20 Bq per nmol) for 30 min at 37°C in a final volume of 200 μl of 50 mM Tris-HCl buffer (pH 7.4). The reaction was terminated by the addition of cold TCA (10% final concentration). The samples were then placed in an ice bath for 30 min and subsequently centrifuged at 16,000×g for 5 min. The [³H]acetate released into the aqueous phase was measured on a liquid scintillation counter. All assays were performed in duplicate. The enzyme activity of Lp-PLA₂ was expressed as pmol of PAF degraded per min per μl of plasma and PAF-AH activity in HPs and HLs was expressed as pmol of PAF degraded per min per mg of sample protein.

Measurement of CD4 cell counts, viral load, and biochemical markers in blood of HIV-infected patients

CD4 cell counts were automatically calculated using the Tetra One System on the EPICS XL flow cytometer, while viral load was determined using the Versant HIV-1 RNA 3.0 assay (bDNA) as previously described.²⁷

Serum TC, HDL-cholesterol, LDL-cholesterol, triglycerides, AST, ALT, ALP, γ- GT, creatinine, and all the other biochemical parameters were measured using appropriate methods in a Siemens Dimension RxL automatic analyzer, as previously described.²⁷

Statistical analysis

Normality of the variables' distribution was assessed using the Kolmogorov–Smirnov criterion. Normally distributed continuous variables were presented as mean values±standard deviation. Differences in mean values of enzyme-specific activities and biochemical parameters were conducted using the repeated measures ANOVA. Correlations and partial correlations between enzyme specific activities and biochemical parameters were evaluated using the Pearson r coefficient. Linear mixed models were conducted in order to estimate the association between the time-dependent changes of PAF-CPT activity and CD4 cell counts. Results are presented as b and SE. The level of statistical significance was set to 0.05, whereas due to multiple comparisons made by the repeated measures method an emphasis was given for borderline significance that was set up to 0.05<p<0.10, only when the effect size together with the biochemical meaning were of importance. Data were analyzed using a statistical software package, SPSS 18.0 (SPSS Inc., Chicago, IL).

Results

Anthropometric and biochemical characteristics of patients

Anthropometric and biochemical characteristics of patients before and after 1, 3, and 6 months of HAART (tenofovir-DF/emtricitabine/efavirenz) initiation are shown in Table 1.

Table 1.

Anthropometric and Biochemical Characteristics of Patients (n=8) before and after 1, 3, and 6 Months of HAART (Tenofovir-DF/Emtricitabine/Efavirenz) Initiation

Characteristics, lipid profile, and biochemical markers	Baseline (0 months)	1 month	3 months	6 months
Age (years)	48±11	—	—	—
Body mass index (BMI) (kg/m²)	28.2±4.1	28.0±4.2	28.1±4.6	28.0±4.2
CD4 (cells/μl)	277±38	357±36^*	429±75^*	389±56^*
Viral load (copies/ml)	40225±43701	223±209	Under 50	Under 50
Total cholesterol (TC) (mg/dl)	174±22	198±23^*	214±40^*	208±31^*
HDL cholesterol (mg/dl)	35±10	41±8^*	43±10^*	46±12^*
LDL cholesterol (mg/dl)	119±22	133±18	147±39^*	139±24^*
Triglycerides (TG) (mg/dl)	101±39	117±52	116±35	115±59
Oxaloacetic transaminase (AST/GOT) (IU/liter)	29±20	27±11	28±14	29±9
Pyruvic transaminase (ALT/GPT) (IU/liter)	35±38	32±23	32±21	33±15
Alkaline phosphatase (ALP) (IU/liter)	62±19	76±15^*	84±16^*	89±19^*
γ-Glutamyltransferase (γ-GT) (IU/liter)	25±15	40±21^*	43±22^*	46±23^*
Lactate dehydrogenase (LDH) (IU/liter)	181±34	183±26	186±31	194±16
Creatine kinase (CK) (IU/liter)	161±134	198±173	144±85	216±144
Amylase (IU/liter)	68±19	71±18	71±23	66±22
Total bilirubin (IU/liter)	0.62±0.22	0.38±0.10^*	0.41±0.16^*	0.47±0.14
Direct bilirubin (IU/liter)	0.11±0.05	0.07±0.03	0.07±0.04^*	0.10±0.07
Indirect bilirubin (IU/liter)	0.51±0.16	0.30±0.10^*	0.34±0.13^*	0.36±0.12^*
Glucose (mg/dl)	95.9±13.6	105.8±17.9	98.5±14.9	101.9±9.2
Urea nitrogen (mg/dl)	13.1±5.0	13.3±4.8	12.9±3.4	12.6±3.2
Creatinine (mg/dl)	0.91±0.15	0.86±0.18	0.86±0.17	0.89±0.16
K (mmol/liter)	4.2±0.3	4.2±0.3	4.4±0.3	4.5±0.4
Na (mmol/liter)	140±1	141±2	141±1	141±3
Ca(II) (mg/dl)	9.3±0.3	9.3±0.4	9.4±0.2	9.5±0.3
P (mg/dl)	2.8±0.5	2.8±0.4	2.9±0.6	2.9±0.7

p<0.05, as compared to baseline values. Comparison of means was conducted using repeated measures ANOVA, after taking into account for multiple comparisons the Bonferroni rule.

Data are expressed as mean values±standard deviation.

All patients had undetectable viral load (<50 copies/ml) at 3 months, while their CD4 cells counts were significantly increased even after the first month of treatment. Regarding their lipid profile, total LDL and HDL cholesterol were significantly increased while triglycerides remained stable. Concerning the hepatic enzymes ALP and γ-GT mean values were increased while AST remained stable. All other biochemical parameters remained stable with the exception of bilirubin (total, direct, and indirect), which was decreased.

Effect of tenofovir-DF/emtricitabine/efavirenz on PAF basic biosynthetic enzymes in blood cells (HLs, HPs) of HIV-infected patients

The activity of PAF-CPT in HLs, which is the main biosynthetic enzyme of the de novo pathway, was significantly reduced even after the first month (approximately 42%, p=0.003) of treatment in comparison to baseline values. This reduction was more pronounced at 6 months, where an approximate reduction of 53% (p=0.025) was observed in comparison to baseline values (Fig. 1a). However, PAF-CPT specific activity in HPs remained relatively stable for up to 6 months of treatment (Fig. 1c).

FIG. 1.

In vivo effect of tenofovir-DF/emtricitabine/efavirenz on PAF basic biosynthetic enzymes in HIV-infected patients. Specific activities of (a) HLs PAF-CPT, (b) HLs lyso-PAF-AT, (c) HPs PAF-CPT, and (d) HPs lyso-PAF-AT are expressed as mean values (pmol/min/mg protein)±standard deviation in each time point. *p<0.10 compared to baseline and #p<0.10 compared to 1 or 3 months of treatment. HLs, human leukocytes; HPs, human platelets; PAF, platelet-activating factor; PAF-CPT, PAF-cholinephosphotransferase; lyso-PAF-AT, lyso-PAF-acetyltransferase.

Concerning the remodeling pathway, the mean value of lyso-PAF-AT activity in HLs, remained relatively stable at the first month while a tendency for a reduction of approximately 22% was observed at the third month of treatment compared to baseline levels. However, this reduction was not found to be statistically significant (p=0.11). On the other hand, this reduction was more pronounced and statistically significant at 6 months since a significant reduction of approximately 57% (p<0.05) was observed compared to baseline levels (Fig. 1b).

Lyso-PAF-AT activity in HLs at 6 months was also found to be borderline significantly lower when compared to its activity at both 1 and 3 months of treatment; a borderline reduction of approximately 53% (p=0.067) and 45% (p=0.072), respectively (Fig. 1d). However, lyso-PAF-AT specific activity in HPs remained relatively stable for up to 6 months of treatment (Fig. 1d).

Effect of tenofovir-DF/emtricitabine/efavirenz on both isoforms of PAF-acetylhydrolase: PAF-AH in blood cells (HLs, HPs) and LpPLA₂ in plasma of HIV-infected patients

Regarding PAF degradation in blood cells of these patients, PAF-AH specific activity in HLs remained relatively stable at the first and third month while it was found to be borderline reduced (approximately 28%, p=0.07) at the sixth month of treatment compared to baseline PAF-AH activity (Fig. 2a).

FIG. 2.

In vivo effect of tenofovir-DF/emtricitabine/efavirenz HAART regimen on PAF-acetylhydrolase isoforms in HIV-infected patients. Specific activities of PAF-acetylhydrolase isoforms: (a) HLs PAF-AH, (b) HPs PAF-AH, and (c) plasma LpPLA₂ are expressed as mean values (pmol/min/mg protein or pmol/min/μl plasma)±standard deviation in each time point. *p<0.10 compared to baseline specific activity. HLs, human leukocytes; HPs, human platelets; PAF, platelet-activating factor; PAF-AH, PAF-acetyl-hydrolase; LpPLA₂, lipoprotein-associated phospholipase A₂ (also known as plasma-PAF-AH).

PAF-AH specific activity in HPs was also found to be borderline reduced even from the first month of treatment (approximately 28%, p=0.09) and remained in borderline lower levels than those in baseline at 6 months (approximately 22%, p=0.09) (Fig. 2b).

In contrast, Lp-PLA₂ (plasma isoform) specific activity remained relatively stable during treatment (Fig. 2c).

Correlations within PAF metabolic enzymes and with other biochemical parameters

Pearson correlation coefficients within PAF metabolic enzymes as well as between them and other baseline biochemical parameters are presented in Table 2.

Table 2.

Pearson Correlation Coefficients Within Platelet-Activating Factor (PAF) Metabolic Enzymes as Well as Between PAF Metabolic Enzymes and Other Baseline Biochemical Parameters

	PAF-CPT of HLs	Lyso-PAF-AT of HLs	PAF-AH of HLs	LpPLA₂
PAF-AH of HLs	0.84	−0.05	—	−0.04
	p=0.01^*	p=0.90		p=0.92
PAF-CPT of HPs	0.33	−0.06	0.62	−0.42
	p=0.41	p=0.87	p=0.10^*	p=0.30
Total cholesterol	−0.23	0.88	−0.08	0.55
	p=0.57	p=0.004^**	p=0.83	p=0.15
LDL	−0.17	0.90	0.04	0.45
	p=0.67	p=0.002^**	p=0.91	p=0.25
Triglycerides	0.46	−0.02	0.34	0.64
	p=0.24	p=0.95	p=0.40	p=0.08
Glucose	0.27	−0.12	−0.09	0.65
	p=0.51	p=0.77	p=0.82	p=0.08^*
BMI	0.66	−0.12	0.88	−0.14
	p=0.10^*	p=0.76	p=0.004^**	p=0.73
CD4	−0.71	−0.17	−0.29	−0.84
	p=0.07	p=0.67	p=0.48	p=0.009^**
Viral load	−0.06	0.06	−0.48	0.66
	p=0.88	p=0.88	p=0.22	p=0.07^*

*,**

Signify statistically significant correlations with p<0.1 and p<0.005, respectively.

PAF-AH and PAF-CPT activities in HLs were positively correlated with BMI at baseline. The PAF-AH activity–BMI correlation was also observed at 6 months (r=0.89, p=0.003) while the PAF-CPT activity–BMI correlation was observed at 1 month (r=0.699, p=0.05) and borderlined at 3 months (r=0.63, p=0.09) of treatment as well, possibly indicating the influence of treatment.

Concerning correlations within PAF metabolic enzymes, PAF-AH activity in HLs was positively correlated with PAF-CPT activity in HLs, a correlation that was also observed at 1 (r=0.79, p=0.01) and 3 months (r=0.74, p=0.03), and with PAF-CPT activity in HPs, a borderline correlation was also observed at 3 months (r=0.62, p=0.09). The positive correlation between PAF-AH activity and PAF-CPT activity in HLs at baseline remained unaffected even after adjusting separately for CD4 cell counts, viral load, age, BMI, lyso-PAF-AT of HLs, and LpPLA₂ (p<0.10, in each case). However, no significant correlation was observed between baseline PAF-AH activity in HLs and PAF-CPT activity in HLs when age and BMI were both taken into account (p>0.10).

Regarding correlations between PAF metabolic enzymes with biochemical parameters of HIV infection, PAF-CPT activity in HLs and plasma LpPLA₂ activity were inversely correlated with CD4 cell counts at baseline. Moreover, LpPLA₂ activity was also positively correlated with serum viral load at baseline. Moreover, the correlation of PAF-CPT activity in HLs with CD4 cell counts was diminished after adjusting for BMI, age, triglycerides, and HDL-C (p>0.10). The correlation between LpPLA₂ activity and CD4 cell counts was not significant after adjusting for BMI, age, and viral load (p>0.10). The correlation between LpPLA₂ activity and viral load was lost after adjusting for CD4 cell counts and age (p>0.10). No significant correlations were observed in other time points, which is probably due to the direct treatment effect on CD4 cell counts and serum viral load.

Concerning the correlation of PAF metabolic enzymes with other biochemical parameters, lyso-PAF-AT activity in HLs at baseline was positively correlated with total cholesterol (r=0.88, p=0.004) and LDL cholesterol (r=0.90, p=0.002). Moreover, LpPLA₂ activity was found to be borderline positively correlated with glucose (r=0.65, p=0.08) and triglycerides (r=0.64, p=0.08) at baseline, borderline correlations that were also present at the first month (r=0.62, p=0.10) for glucose and at 6 months for triglycerides (r=0.70, p=0.05).

In addition, viral load and CD4 cell counts were borderline inversely correlated, after adjusting for PAF-CPT activity in HLs (r=–0.76, p=0.07) or for BMI (r=–0.81, p=0.02). Furthermore, lyso-PAF-AT activity in HLs was positively correlated with lyso-PAF-AT activity in HPs at 3 (r=0.91, p=0.002) and at 6 months (r=0.78, p=0.02) of treatment, PAF-AH activity in HLs was found to be borderline positively correlated with that in HPs (r=0.68, p=0.06) at 3 months, and Lp-PLA₂ activity was positively correlated with total cholesterol (r=0.74, p=0.03) and LDL-C (r=0.74, p=0.03) at 6 months of treatment.

Time dependence between PAF-CPT of HLs and CD4 cell counts

To evaluate the association between the treatment-dependent modulation and the distribution of biochemical parameters, repeated measures analysis for repeated measured covariates (using mixed models) was performed having as the dependent outcome CD4 cell counts and as the independent variables viral load and PAF-CPT activity in HLs, at baseline, 1, 3, and 6 months of treatment. The analysis showed that CD4 cell counts were positively associated with viral load (2×10⁻³±4×10⁻⁴, p=0.007), while a highly significant interaction was observed between PAF-CPT activity in HLs, viral load, and CD4 cell counts (–0.12±0.004, p=0.01). Thus, the analysis was further stratified by viral load level (i.e., below or above the threshold of 50). It was observed that PAF-CPT activity in HLs was inversely associated with CD4 cell counts (–151.6±61.71, p=0.05) among subjects with detectable viral load, whereas no association was observed between PAF-CPT activity in HLs and CD4 cell counts (–1.71±51.10, p=0.97) among these subjects.

Discussion

PAF seems to play a significant role in the pathogenesis of several AIDS manifestations.^15
–17 In such situations, induced PAF synthesis evokes inflammatory conditions that favor HIV replication, monocytic cell recruitment, and increased vascular permeability, events that take place in the initiation and progression of HIV-related disorders.^15

–18 PAF inhibitors have shown promising results in these AIDS manifestations, while several of them have exhibited not only anti-PAF but also anti-HIV activities.¹⁸

We have previously indicated that PAF metabolism is altered in vivo in naive asymptomatic HIV-infected patients.²² We have found an increase in both PAF biosynthetic routes accompanied by an initial increase of PAF degradation as a host response that, subsequently, gradually decreased during progression of HIV infection. On the other hand, we have indicated that apart from their general anti-HIV activities, some antiretroviral drugs, such as tenofovir-DF, exhibit a potent inhibitory effect against PAF biological activities and also inhibit its basic biosynthetic enzymes, PAF-CPT and lyso-PAF-AT, when added in the cell medium of human mesangial cells.¹⁹ Moreover, preliminary in vivo results have shown that in three naive patients treated for a period of 1 month with tenofovir-DF/emtricitabine/efavirenz, a significant reduction of the specific activity of PAF-CPT in HLs was also observed, while lyso-PAF-AT of HLs and Lp-PLA₂ were unaffected.¹⁹

In the present study, we have further investigated in HIV-infected patients the in vivo effect of the aforementioned HAART regimen on PAF metabolism. More specifically, it is the first report indicating that during treatment with tenofovir-DF/emtricitabine /efavirenz in naive, male HIV-infected patients for up to 6 months, the basic enzyme activities of both PAF biosynthetic pathways were found to be reduced in HLs but stable in HPs compared to their baseline levels before HAART initiation.

Tenofovir-DF/emtricitabine/efavirenz successfully suppressed viral load to undetectable levels (<50 copies/ml) in all patients at 3 months and CD4 cell count was significantly increased even from the first month.

Concerning PAF metabolism, PAF-CPT activity of HLs was reduced even from the first month of treatment compared to baseline, a result that accords with our previously reported findings.¹⁹ Furthermore, this reduction was sustained and it was more intense at 6 months of treatment, reaching the previously reported levels in healthy volunteers.²² Regarding the remodeling PAF biosynthesis in HLs, lyso-PAF-AT activity in these cells remained relatively stable at the first month of treatment compared to its levels at baseline, a result that is also accordance with our previously reported findings.¹⁹ A tendency for reduction of this enzyme's activity began at the third month of treatment, after PAF-CPT of HLs and viral load were reduced, while it was statistically significantly reduced at 6 months, where it reached the previously reported levels in healthy volunteers.²² Moreover, lyso-PAF-AT activity of HLs at 6 months of treatment was found to be borderline lower when compared to that at both 1 and 3 months of treatment. Since this enzyme is implicated in inflammatory procedures²⁸ its significant reduction at 6 months of treatment with tenofovir-DF/emtricitabine/efavirenz seems to further support the protective effects of this HAART regimen toward PAF-related inflammatory HIV manifestations.

On the other hand, lyso-PAF-AT and PAF-CPT activities of HPs remained relatively stable during treatment compared to their baseline levels. These enzyme activities were at least one order of magnitude lower than that of HLs at baseline. Even though lyso-PAF-AT and PAF-CPT activities of HLs were reduced after 6 months of treatment, they still remained higher than the ones in HPs. Therefore, it seems that PAF biosynthetic enzyme activities in HLs contribute more to PAF production during HIV infection and are more susceptible to reduction by HAART treatment compared to the ones in HPs. A plausible explanation might be the regulation of genes encoding for these enzymes since HLs have a nucleus in contrast to mature HPs. However, the amino acid sequence of PAF-CPT has not yet been characterized and the gene responsible for its expression has not been identified, while cloning and characterization of lyso-PAF-AT have just recently been achieved.²⁸ Subsequently, more studies are needed to confirm the above observations and clarify the relative contribution of these enzyme activities in different blood cell types in various pathological conditions before and after treatment.

In systemic inflammatory conditions such as AIDS and atherosclerosis, the activity of LpPLA₂ was found to be increased probably as a response to pathologically increased PAF levels and oxidized phospholipids with PAF-like activity.^22,29,30 We have previously indicated that increased LpPLA₂ activity is observed in naive patients during progression of HIV infection.²² However, results from the present study revealed that LpPLA₂ activity remained relatively stable during HAART treatment. This is in accordance with the study by Klovidhunic et al. in which it is reported that a successful suppression of HIV RNA levels by either a protease inhibitor or the nucleoside analog lamivudine was not accompanied by a lower level of LpPLA₂ activity, suggesting that LpPLA₂ activity may be a sensitive marker of the host response to infection.²⁹

On the other hand, it has been reported for the first time that PAF-AH activities of both HLs and HPs were found to be borderline reduced at 6 months of treatment compared to their baseline levels. These results also suggest that increased PAF biosynthesis in these patients before initiation of treatment is accompanied by increased intracellular PAF-AH activity as a response, while the decline of this enzyme's activity during treatment reflects the reduction of PAF biosynthesis. This is further supported by the fact that PAF-AH activity of HLs was found to be positively correlated with PAF-CPT activities of both HLs and HPs at baseline and at certain time points during treatment. However, the decline of the PAF-AH isoform in blood cells at 6 months was not observed in the LpPLA₂ isoform in plasma, suggesting that the aftereffect of this reduction in Lp-PLA₂ levels might be observed later or that Lp-PLA₂ activity originates not only from blood cells but also from other tissues.

PAF-CPT of HLs and plasma LpPLA₂ activities were found to be inversely correlated with CD4 cell counts at baseline. These results are consistent with our previously reported observations in which a similar inverse correlation between PAF-CPT or LpPLA₂ activity and CD4 cell counts as well as a positive correlation between LpPLA₂ activity and viral load were found in the group of patients with late HIV infection but not in patients with early HIV infection, implying a relation between viral load, PAF-CPT, and CD4 cell counts in the late phase of HIV infection and especially in patients that need HAART initiation.²² Our results also support the idea that the reduction of CD4 cell counts with uncontrolled viremia during progression of HIV infection is related to increased PAF biosynthesis. This is further supported by reports indicating that HIV infection induces PAF synthesis¹⁶ and that PAF participates in the process of progressive immunosuppression inhibiting CD4 cell proliferation^31,32 participating in progressive immunosuppression. Furthermore, during treatment this correlation was absent. Since viral load was simultaneously diminished with the increase of CD4 cell counts and also along with the reduction of PAF-CPT activity of HLs, accompanied by a reduction in lyso-PAF-AT activity, this suggests that the restored activity of PAF biosynthetic enzymes to their normal levels could not affect CD4 cell proliferation during treatment. In addition, the mixed model analysis showed that the increase of CD4 cell counts during treatment is dependent only on the decrease of both viral load and HLs PAF-CPT. As a result, the effect of such HAART regimens in PAF biosynthesis seems to provide favorable outcomes not only for PAF-related inflammatory HIV manifestations but also for HIV-induced immunosuppression.

It should also be mentioned that apart from the above-mentioned changes in PAF metabolism during this HAART treatment, we have also observed some changes in lipid profile and hepatic enzymes, while all other biochemical parameters remained stable during this time period. Therefore it may be that the tenofovir-DF/emtricitabine/efavirenz treatment may reflect the combined favorable effects of this HAART regimen toward both HIV infection and PAF metabolism without affecting other systems. However, more in vivo studies are needed in a larger scale of patients combined with a longer duration of follow-up to clarify the potential implication of HAART-influenced PAF metabolism in favorable effects or side-effects of each one of the HAART regimens usually administrated.

Conclusions

In conclusion, we report for the first time that apart from the expected reduction of viral load and the subsequent restoration of CD4 cell counts in naive HIV patients treated with a HAART regimen containing tenofovir-DF/emtricitabine/efavirenz for a period of 6 months, a significant reduction of both de novo and remodeling PAF biosynthetic routes in leukocytes of these patients was also observed. Treatment also reduced PAF degradation in blood cells, while it did not affect any PAF biosynthetic routes in platelets or plasma LpPLA₂. In addition, a time-dependent correlation between the increase in CD4 cell counts and the decrease in both viral load and HLs PAF-CPT activity seems to occur during treatment. Since PAF is implicated in inflammatory endothelial dysfunction in proatherosclerotic complications and along with other mediators seems to be involved in several HIV-induced manifestations, the in vivo effect of tenofovir-DF/emtricitabine/efavirenz on PAF metabolism may provide additional data in the field of HIV therapeutics in order to optimize antiretroviral treatment.

Footnotes

Acknowledgments

This work was supported by a grant from the Hellenic Society for Research, Study, and Education in Infectious Diseases. In addition, the authors would like to thank Dr. Demosthenes B. Panagiotakos (Associate Professor in Biostatistics-Nutrition Epidemiology at the Department of Nutrition and Dietetics of the Harokopio University of Athens in Greece) for his advice concerning the statistical analysis.

Authors' contributions: M.C.: conceived of the study and participated in its design and the coordination and management of patients and the review of the manuscript. A.B.T.: conceived of the study, participated in its design and coordination, carried out the in vivo and in vitro assays including blood samples management, separation of cells and plasma from patients' blood, PAF metabolic enzyme assays, TLC, biological test in rabbit platelets, and analysis/statistics of data, and drafted and reviewed the manuscript. N.M.: participated in the design of the study and in the management of patients. N.T.: participated in the design of the study. VDP: helped in the in vivo and in vitro assays including blood samples management, separation of cells and plasma from patients' blood, PAF metabolic enzyme assays, TLC, and biological test in rabbit platelets. E.F.: helped in the analysis/statistics of the data and in the draft and review of the manuscript. S.A.: participated in the design of the study and helped in the final review of the manuscript. P.G.: participated in the design of the study. C.A.D.: conceived the study, participated in its design and coordination, and helped to draft and review of the manuscript. M.C.L.: conceived of the study and participated in its design and coordination and review of the manuscript. All authors have read and approved the final manuscript.

Author Disclosure Statement

No competing financial interests exist.

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Effects of HAART on Platelet-Activating Factor Metabolism in Naive HIV-Infected Patients I: Study of the Tenofovir-DF/Emtricitabine/Efavirenz HAART Regimen

Abstract

Introduction

Materials and Methods

Materials and instruments

Study design

Isolation of plasma, leukocytes, and platelets from blood samples

Isolation of plasma

Isolation of human platelets (HPs)

Isolation of human leukocytes (HLs)

Assays

PAF-CPT activity assays

Lyso-PAF-AT activity assays

Determination of produced PAF and biosynthetic enzymes (PAF-CPT and lyso-PAF-AT) activities

PAF-acetylhydrolase activity assays

Measurement of CD4 cell counts, viral load, and biochemical markers in blood of HIV-infected patients

Statistical analysis

Results

Anthropometric and biochemical characteristics of patients

Effect of tenofovir-DF/emtricitabine/efavirenz on PAF basic biosynthetic enzymes in blood cells (HLs, HPs) of HIV-infected patients

Effect of tenofovir-DF/emtricitabine/efavirenz on both isoforms of PAF-acetylhydrolase: PAF-AH in blood cells (HLs, HPs) and LpPLA2 in plasma of HIV-infected patients

Correlations within PAF metabolic enzymes and with other biochemical parameters

Time dependence between PAF-CPT of HLs and CD4 cell counts

Discussion

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

References

Effect of tenofovir-DF/emtricitabine/efavirenz on both isoforms of PAF-acetylhydrolase: PAF-AH in blood cells (HLs, HPs) and LpPLA₂ in plasma of HIV-infected patients