Background and purpose: Transgenesis of human paraoxonase 1 (PON1), a HDL‐associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E‐deficient (apoE −/− ) mice with advanced atherosclerosis. Experimental approach: ApoE −/− mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non‐transfected apoE −/− (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. Key results: PON1 gene transfer raised total PON1 serum activity 13‐15 fold during the 3‐week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque‐free thoracic aorta rings from apoE −/− mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86±2%), ATP (90±2%) or UTP (83±3%). In contrast, in plaque‐bearing segments amplitude (55±7%, 68±8%, 52±8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE −/− SMCs released less intracellular calcium than WT upon sarco‐endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. Conclusions and implications: These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca 2+ homeostasis in segments with pre‐existing atherosclerosis, independently of an effect on plaque size. British Journal of Pharmacology (2008) 153 , 508–516; doi: 10.1038/sj.bjp.0707585 ; published online 3 December 2007
Oxidative stress is associated with human immunodeficiency virus (HIV) infection. Paraoxonase-1 (PON1) is an antioxidant enzyme that is bound to high-density lipoproteins (HDLs). We evaluated whether PON1 gene haplotypes influence the metabolic disturbances, presence of subclinical atherosclerosis, and virologic outcome associated with the infection.DNA from blood samples collected from 234 HIV-infected patients and 633 healthy control subjects had single-nucleotide polymorphisms of PON1(192), PON1(55), PON1(-162), PON1(-832), PON1(-909), PON1(-1076), and PON1(-1741) analyzed using the Iplex Gold MassArray method. Subsequently, the influence of these single-nucleotide polymorphisms on measured biochemical and clinical variables was assessed.We observed significant differences in the haplotype distribution between the control subjects and the HIV-infected patients. Haplotype H10 (GTCCGTC) was more prevalent in the HIV-infected patients (6.41% vs 0.64%; P < .001), and haplotype H5 (GACCGTC) was less prevalent in HIV-infected patients (27.7% vs 42.9%; P = .001). In HIV-infected patients, haplotype H7 (AATTCCT) was associated with better CD4(+) cell count recovery, higher levels of HDL cholesterol (P = .048) and apolipoprotein A-I (P = .019), lower levels of triglycerides (P = .004), and lower rates of subclinical arteriosclerosis (P < .001).PON1 haplotypes segregate with HIV infection, HDL metabolism, the presence of subclinical atherosclerosis, and CD4(+) cell recovery after treatment.
We investigated the influence of the HIV infection on serum paraoxonase-3 (PON3) concentration and assessed the relationships with lipoprotein-associated abnormalities, immunological response, and accelerated atherosclerosis. We studied 207 HIV-infected patients and 385 healthy volunteers. Serum PON3 was determined by in-house ELISA, and PON3 distribution in lipoproteins was investigated by fast-performance liquid chromatography (FPLC). Polymorphisms of the PON3 promoter were analyzed by the Iplex Gold MassArrayTM method. PON3 concentrations were increased (about three times) in HIV-infected patients with respect to controls (P < 0.001) and were inversely correlated with oxidized LDL levels (P = 0.038). Long-term use of nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy was associated with a decrease of PON3 concentrations. In a multivariate linear regression analysis, these relationships were still strong when the main confounding covariates were considered. PON3 was mainly found in HDL in HIV-infected patients, but a substantial amount of the protein was detected in LDL particles. This study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and their treatment with NNRTI. Long-term, prospective studies are needed to confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker. We investigated the influence of the HIV infection on serum paraoxonase-3 (PON3) concentration and assessed the relationships with lipoprotein-associated abnormalities, immunological response, and accelerated atherosclerosis. We studied 207 HIV-infected patients and 385 healthy volunteers. Serum PON3 was determined by in-house ELISA, and PON3 distribution in lipoproteins was investigated by fast-performance liquid chromatography (FPLC). Polymorphisms of the PON3 promoter were analyzed by the Iplex Gold MassArrayTM method. PON3 concentrations were increased (about three times) in HIV-infected patients with respect to controls (P < 0.001) and were inversely correlated with oxidized LDL levels (P = 0.038). Long-term use of nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy was associated with a decrease of PON3 concentrations. In a multivariate linear regression analysis, these relationships were still strong when the main confounding covariates were considered. PON3 was mainly found in HDL in HIV-infected patients, but a substantial amount of the protein was detected in LDL particles. This study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and their treatment with NNRTI. Long-term, prospective studies are needed to confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker. Human immunodeficiency virus (HIV)-infected patients often develop long-term metabolic alterations and concomitant atherosclerosis (1.Boyd M. Reiss P. The long-term consequences of antiretroviral therapy: a review.J. HIV Ther. 2006; 11: 26-35PubMed Google Scholar, 2.Bergersen B.M. Cardiovascular risk in patients with HIV infection: impact of antiretroviral therapy.Drugs. 2006; 66: 1971-1987Crossref PubMed Scopus (56) Google Scholar). This association has acquired clinical relevance since the introduction of effective therapeutic measures that have modified HIV infection to a chronic disease and as the consequences of metabolic derangements become more evident over time. In the course of HIV infection, there are several key changes in lipoprotein metabolism, including increased lipid peroxidation, hypertriglyceridemia, and low high-density lipoprotein (HDL) concentration (3.Rose H. Woolley I. Hoy J. Dart A. Bryant B. Mijch A. Sviridov D. HIV infection and high-density lipoprotein: the effect of disease vs. the effect of treatment.Metabolism. 2006; 55: 90-95Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Among them, changes in HDL are particularly relevant, as HIV-infected patients with higher HDL-cholesterol concentrations appear to have a better HIV disease course than those with lower HDL concentrations (4.Alonso-Villaverde C. Segués T. Coll-Crespo B. Pérez-Bernalte R. Rabassa A. Gomila M. Parra S. Gozález-Esteban M.A. Jiménez-Expósito M.J. Masana L. High-density lipoprotein concentrations relate to the clinical course of HIV viral load in patients undergoing antiretroviral therapy.AIDS. 2003; 17: 1173-1178Crossref PubMed Scopus (30) Google Scholar). In addition, we have previously documented that serum paraoxonase-1 (PON1) activity and concentration are influenced by HIV infection (5.Parra S. Alonso-Villaverde C. Coll B. Ferré N. Marsillach J. Aragonès G. Mackness M. Mackness B. Masana L. Joven J. et al.Serum paraoxonase-1 activity and concentration are influenced by human immunodeficiency virus infection.Atherosclerosis. 2007; 194: 175-181Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and that PON1 gene polymorphisms are related to the presence of subclinical atherosclerosis and CD4+ T-cell recovery following treatment (6.Parra S. Marsillach J. Aragonès G. Beltrán R. Montero M. Coll B. Mackness B. Mackness M. Alonso-Villaverde C. Joven J. et al.Association of paraoxonase-1 gene haplotypes with the immunologic outcome of and metabolic disturbances and atherosclerosis in HIV-infected patients.J. Infect. Dis. 2010; 201: 627-634Crossref PubMed Scopus (23) Google Scholar). The PON enzyme family comprises three members, PON1, PON2, and PON3, whose genes are located adjacent to each other on chromosome 7q21-22 (7.Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (578) Google Scholar). In mammals, the PON1 and PON3 genes are expressed in many cell types (8.Rodríguez-Sanabria F. Rull A. Beltrán-Debón R. Aragonès G. Camps J. Mackness B. Mackness M. Joven J. Tissue distribution and expression of paraoxonases and chemokines in mouse: the ubiquitous and joint localisation suggest a systemic and coordinated role.J. Mol. Histol. 2010; 41: 379-386Crossref PubMed Scopus (48) Google Scholar), and their protein products are found in the bloodstream bound to HDL (9.Deakin S.P. Bioletto S. Bochaton-Piallat M.L. James R.W. HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress.Free Radic. Biol. Med. 2011; 50: 102-109Crossref PubMed Scopus (75) Google Scholar). Conversely, PON2 is an intracellular enzyme that is not found in the bloodstream (10.Ng C.J. Wadleigh D.J. Gangopadhyay A. Hama S. Grijalva V.R. Navab M. Fogelman A.M. Reddy S.T. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.J. Biol. Chem. 2001; 276: 44444-44449Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). All these enzymes are able to delay low-density lipoprotein (LDL) oxidation and cellular oxidative stress (11.Camps J. Marsillach J. Joven J. The paraoxonases: role in human diseases and methodological difficulties in measurement.Crit. Rev. Clin. Lab. Sci. 2009; 46: 83-106Crossref PubMed Scopus (207) Google Scholar). In addition, data obtained from a variety of mouse models of atherosclerosis have consistently shown that human PON1, 2, or 3 expression inhibits or reverses the development of atherosclerosis via mechanisms involving the reduction of oxidative stress, the promotion of cholesterol efflux from macrophages, and the normalization of vascular endothelium function (12.Mackness B. Quarck R. Verreth W. Mackness M. Holvoet P. Human paraoxonase-1 overexpression inhibits atherosclerosis in a mouse model of metabolic syndrome.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1545-1550Crossref PubMed Scopus (145) Google Scholar, 13.Ng C.J. Hama S.Y. Bourquard N. Navab M. Reddy S.T. Adenovirus mediated expression of human paraoxonase 2 protects against the development of atherosclerosis in apolipoprotein E-deficient mice.Mol. Genet. Metab. 2006; 89: 368-373Crossref PubMed Scopus (70) Google Scholar, 14.Shih D.M. Xia Y.R. Wang X.P. Wang S.S. Bourquard N. Fogelman A.M. Lusis A.J. Reddy S.T. Decreased obesity and atherosclerosis in human paraoxonase 3 transgenic mice.Circ. Res. 2007; 100: 1200-1207Crossref PubMed Scopus (84) Google Scholar, 15.Ng C.J. Bourquard N. Hama S.Y. Shih D. Grijalva V.R. Navab M. Fogelman A.M. Reddy S.T. Adenovirus-mediated expression of human paraoxonase 3 protects against the progression of atherosclerosis in apolipoprotein E-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1368-1374Crossref PubMed Scopus (48) Google Scholar). Moreover, recent studies showed that PON2 expression is increased in cultured hematopoietic cells and mouse thymocytes after HIV-1 infection (16.Yuan J. Devarajan A. Moya-Castro R. Zhang M. Evans S. Bourquard N. Dias P. Lacout C. Vainchenker W. Reddy S.T. et al.Putative innate immunity of antiatherogenic paraoxonase-2 via STAT5 signal transduction in HIV-1 infection of hematopoietic TF-1 cells and in SCID-hu mice.J. Stem Cells. 2010; 5: 43-48PubMed Google Scholar). The PON family also plays a role in innate immunity and can prevent bacterial infection (17.Camps J. Pujol I. Ballester F. Joven J. Simó J.M. Paraoxonases as potential antibiofilm agents: their relationship with quorum-sensing signals in gram-negative bacteria.Antimicrob. Agents Chemother. 2011; 55: 1325-1331Crossref PubMed Scopus (55) Google Scholar). Although knowledge on PON1 and PON2 structure and function is rapidly expanding, data about the PON3 protein remain elusive. Its gene was identified in 1996 when Primo-Parmo et al. (7.Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (578) Google Scholar) detected a large number of cDNA sequences in the Genome Data Base with significant similarity to, but not identical with, human PON1. The percentage identity among human PON1, PON2, and PON3 genes is high (about 70%), and the genes are believed to derive from a common precursor (11.Camps J. Marsillach J. Joven J. The paraoxonases: role in human diseases and methodological difficulties in measurement.Crit. Rev. Clin. Lab. Sci. 2009; 46: 83-106Crossref PubMed Scopus (207) Google Scholar). Clinical research on PON3 has been hampered by the lack of methods for measurement, but we recently described a high-throughput, reliable enzyme-linked immunosorbent assay (ELISA) to analyze PON3 concentration in human serum (18.Aragonès G. Guardiola M. Barreda M. Marsillach J. Beltran-Debon R. Rull A. Mackness B. Mackness M. Joven J. Simó J.M. et al.Measurement of serum paraoxonase-3 concentration: method evaluation, reference values and influence of genotypes in a population-based study.J. Lipid Res. 2011; 52: 1055-1061Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The main objective of the present study was to investigate whether serum PON3 concentration may provide new information to improve our understanding of metabolic complications associated with HIV infection. From among the HIV-infected patients attending our clinic, 207 (139 men, 68 women; mean age, 38 years; range, 22-66 years) accepted an invitation to participate in the present study. Of these patients, 122 were coinfected by the hepatitis C virus (HCV). All patients were undergoing antiretroviral therapy with protease inhibitor (PI)-based or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based schemes. The antiretroviral adjuvant drugs were zidovudine, stavudine, didanosine, or lamivudine. The exclusion criteria were age under 18 years, or renal function impairment defined as creatinine levels higher than 106 μmol/l, or having an AIDS-related opportunistic disease at the time of the study. Twenty-five patients had subcutaneous lipoatrophy, defined as the presence of hollow cheeks, prominent superficial veins in the limbs, or flattening of the buttocks (19.Martínez E. Mocroft A. García-Viejo M.A. Pérez-Cuevas J.B. Blanco J.L. Mallolas J. Bianchi L. Conget I. Blanch J. Phillips A. et al.Risk of lipodystrophy in HIV-1 infected patients treated with protease inhibitors: a prospective cohort study.Lancet. 2001; 357: 592-598Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Carotid and femoral ultrasound measurements were performed in 178 patients and the intima-media thickness (IMT) was measured as an estimate of the presence of subclinical atherosclerosis, as previously described (20.Alonso-Villaverde C. Coll B. Parra S. Montero M. Calvo N. Tous M. Joven J. Masana L. Atherosclerosis in patients infected with HIV is influenced by a mutant monocyte chemoattractant protein-1 allele.Circulation. 2004; 110: 2204-2209Crossref PubMed Scopus (100) Google Scholar). Patients were considered to have subclinical atherosclerosis when IMT was ≥ 0.8 mm or when an atheromatous plaque was seen in the analyzed areas of the arteries. The main clinical characteristics of these patients are summarized in supplementary Table I. The control group consisted of 385 healthy volunteers (153 men, 232 women; mean age, 47 years; range, 19-75 years) who participated in an ongoing epidemiological study conducted in our geographic area, the details of which have been previously reported (21.Ferré N. Camps J. Fernández-Ballart J. Arija V. Murphy M.M. Ceruelo S. Biarnés E. Vilella E. Tous M. Joven J. Regulation of serum paraoxonase activity by genetic, nutritional, and lifestyle factors in the general population.Clin. Chem. 2003; 49: 1491-1497Crossref PubMed Scopus (142) Google Scholar). All the volunteers had been invited to attend a clinical examination and to provide a fasting blood sample. There was no clinical or analytical evidence of renal insufficiency, liver damage, neoplasia, or neurological disorders. A fasting venous blood sample was obtained from all the participants. CD4+ T cells and CD8+ T cells were analyzed immediately, and serum, plasma, and leukocytes were stored at −80°C in our biological sample bank until the other measurements were performed. We employed independent aliquots that were never thawed before this investigation, although participants in this study partially coincided with those reported in previous investigations (5.Parra S. Alonso-Villaverde C. Coll B. Ferré N. Marsillach J. Aragonès G. Mackness M. Mackness B. Masana L. Joven J. et al.Serum paraoxonase-1 activity and concentration are influenced by human immunodeficiency virus infection.Atherosclerosis. 2007; 194: 175-181Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6.Parra S. Marsillach J. Aragonès G. Beltrán R. Montero M. Coll B. Mackness B. Mackness M. Alonso-Villaverde C. Joven J. et al.Association of paraoxonase-1 gene haplotypes with the immunologic outcome of and metabolic disturbances and atherosclerosis in HIV-infected patients.J. Infect. Dis. 2010; 201: 627-634Crossref PubMed Scopus (23) Google Scholar). All the participants provided fully informed consent to participation in the study on the understanding that anonymity of all data is guaranteed. The study was approved by the Institutional Review Board of the Hospital Universitari de Sant Joan de Reus. Serum PON3 concentrations were determined by in-house ELISA using rabbit polyclonal antibodies generated against a synthetic peptide with a sequence specific to mature PON3. Details of this method have been previously reported (18.Aragonès G. Guardiola M. Barreda M. Marsillach J. Beltran-Debon R. Rull A. Mackness B. Mackness M. Joven J. Simó J.M. et al.Measurement of serum paraoxonase-3 concentration: method evaluation, reference values and influence of genotypes in a population-based study.J. Lipid Res. 2011; 52: 1055-1061Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 22.Marsillach J. Mackness B. Mackness M. Riu F. Beltrán R. Joven J. Camps J. Immunochemical analysis of paraoxonases-1, 2, and 3 expression in normal mouse tissues.Free Radic. Biol. Med. 2008; 45: 146-157Crossref PubMed Scopus (149) Google Scholar). Plasma viral load was measured with the COBAS® TaqMan® HIV-1 assay (Roche, Basel, Switzerland), and CD4+ T-cell and CD8+ T-cell counts were measured by flow cytometry (Coulter Epics XL-MLC, Beckman Coulter, Fullerton, CA). Antibodies against HCV, serum β-2-microglobulin [a marker of lymphocyte destruction and progression of HIV-infection (23.Savès M. Morlat P. Chêne G. Peuchant E. Pellegrin I. Bonnet F. Bernard N. Lacoste D. Salamon R. Beylot J. Prognostic value of plasma markers of immune activation in patients with advanced HIV disease treated by combination antiretroviral therapy.Clin. Immunol. 2001; 99: 347-352Crossref PubMed Scopus (23) Google Scholar)], and serum cholesterol, triglycerides, HDL-cholesterol, and apolipoprotein (apo)A-I were measured in an automated analyzer (UniCelTM DxI 800, Beckman Coulter, Fullerton, CA). Oxidized LDL levels were measured by ELISA (Mercodia, Uppsala, Sweden). PON3 distribution in lipoproteins was assessed by FPLC (Bio-Rad BioLogic DuoFlow 10 system, Bio-Rad Laboratories, Hercules, CA). Sera from three HIV-infected patients and three noninfected participants were pooled separately. To maximize the possible differences between groups, sera from the HIV-infected patients were chosen to have a PON3 concentration > 20 mg/l. Two-hundred microliters from each pool were injected into a Superose 6/300 GL column (GE Healthcare Europe, Glattbrugg, Switzerland), and 500 μl fractions were collected. Cholesterol, triglycerides, and PON3 in each fraction were measured as described. Genomic DNA was obtained from leukocytes (Puregene DNA Isolation reagent set, Gentra Systems, Minneapolis, MN). Selected single nucleotide polymorphisms (SNP) of the PON3 promoter were analyzed by the Iplex Gold MassArrayTM method (Sequenom, San Diego, CA) at the Spanish National Genotyping Center (Centro Nacional de Genotipado, Universitat Pompeu Fabra, Barcelona, Spain). The normality of distributions was determined with the Kolmogorov-Smirnov test. Differences between two groups were assessed with the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Differences between multiple groups were analyzed by the Kruskal-Wallis test. Pearson or Spearman correlation coefficients were used to evaluate the degree of association between variables. Each SNP was tested for Hardy-Weinberg equilibrium using Haploview 4.0 software (24.Barrett J.C. Fry B. Maller J. Daly M.J. Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (12023) Google Scholar). Estimates of linkage disequilibrium between SNPs were calculated using Fisher's test. Diagnostic accuracy for the measurement of serum PON3 concentration was calculated with ROC analysis (25.Zweig M.H. Campbell G. Receiver-operating characteristics (ROC) plots: a fundamental evaluation tool in clinical medicine.Clin. Chem. 1993; 39: 561-577Crossref PubMed Scopus (5298) Google Scholar). A multiple linear regression model was fitted to evaluate the factors that were independently associated with PON3 concentrations in HIV-infected patients. Results are shown as means and SD (parametric) or as medians and 95% confidence interval (CI; nonparametric). The SPSS 18.0 package was used for all statistical calculations. Serum PON3 concentrations were significantly increased in HIV-infected patients with respect to the control group [5.5 (1.2-10.8) versus 1.8 (1.0-2.5) mg/l, respectively; P < 0.001; Fig. 1A]. The results of the ROC analysis for serum PON3 concentration measurement are shown in Fig. 1B. The area under the curve (AUC) was 0.94 (95% CI: 0.92-0.97; P < 0.001), which highlights the remarkable differences in serum PON3 concentrations between patients and controls. We observed a significant inverse relationship (r =−0.147; P = 0.038) between serum PON3 concentration and oxidized LDL levels (Fig. 1C) in HIV-infected patients but not in the control group (r = 0.024; P = 0.786). HIV-infected patients were characterized by raised serum triglyceride concentration and decreased cholesterol values in HDL and LDL (Table 1); there were no significant associations among serum PON3 concentrations, cholesterol, and triglycerides (supplementary Table II).TABLE 1Selected biochemical variables in the control group and in HIV-infected patientsParameterControl group(n = 385)HIV-infected patients(n = 207)PCholesterol (mmol/l)5.28 (0.98)4.89 (1.23)<0.001Triglycerides (mmol/l)1.1 (0.5-2.6)1.5 (0.6-8.5)<0.001HDL-cholesterol (mmol/l)1.48 (0.39)1.18 (0.45)<0.001LDL-cholesterol (mmol/l)3.20 (0.95)2.75 (0.96)<0.001Apolipoprotein A-I (g/l)1.69 (0.28)1.38 (0.31)<0.001Oxidized LDL (U/l)84.5 (81.8-88.7)81.5 (40.5-145.9)0.951Results are presented as means and SD in parentheses (parametric) or as medians and 95% CI in parenthesis (nonparametric). Open table in a new tab Results are presented as means and SD in parentheses (parametric) or as medians and 95% CI in parenthesis (nonparametric). In noninfected participants, PON3 immunoreactivity was observed almost exclusively in HDL fractions. However, in the HIV-infected pool, a substantial amount of this protein eluted with the smallest HDL and with LDL particles (Fig. 2). The frequency distributions of the selected PON3 promoter gene polymorphisms are shown in Table 2. There were no significant differences between control subjects and HIV-infected patients. These polymorphisms moderately influenced serum PON3 concentrations in the control subjects but not in the patient group.TABLE 2.Distribution of PON3 genotypes in the control group and HIV-infected patientsGenotype frequency (%)PON3 (mg/l)PolymorphismControlHIVControlHIVPON3-567aPolymorphisms associated with significant changes (P < 0.05) in the control group but not in HIV-infected patients.CC59.861.71.83 (0.45)5.97 (0.21)CT36.432.11.68 (0.41)6.19 (0.31)TT3.86.31.56 (0.28)5.51 (0.55)PON3-665aPolymorphisms associated with significant changes (P < 0.05) in the control group but not in HIV-infected patients.AA59.661.71.83 (0.45)5.97 (0.21)AG36.632.11.68 (0.40)6.19 (0.31)GG3.86.31.56 (0.28)5.51 (0.55)PON3-746aPolymorphisms associated with significant changes (P < 0.05) in the control group but not in HIV-infected patients.CC59.761.51.83 (0.44)5.98 (0.21)CT36.531.81.67 (0.41)6.21 (0.32)TT3.86.61.56 (0.27)5.45 (0.52)PON3-4105GG61.764.81.82 (0.45)5.95 (0.20)GA35.330.01.68 (0.40)6.18 (0.32)AA3.05.21.56 (0.32)5.89 (0.66)PON3-4970TT62.764.51.82 (0.45)5.99 (0.20)TG34.830.71.69 (0.39)6.14 (0.32)GG2.54.91.51 (0.31)5.54 (0.60)PON3-4984AA62.464.61.81 (0.46)5.97 (0.20)AG34.830.51.69 (0.39)6.08 (0.32)GG2.84.91.51 (0.31)5.54 (0.60)a Polymorphisms associated with significant changes (P < 0.05) in the control group but not in HIV-infected patients. Open table in a new tab All PON3 promoter polymorphisms were strongly linked in a single haplotype, and we did not observe any significant differences between patients and controls (supplementary Fig. I). Coinfection with HCV was associated with a significantly higher PON3 concentration [5.8 (2.6-11.1) versus 4.5 (2.4-11.6) mg/l, respectively; P = 0.024]. There were not any significant associations between serum PON3 concentrations and CD4+ T-cell and CD8+ T-cell counts, the CD4+/CD8+ ratio, or the plasma HIV-1 viral load (supplementary Table III). There was a significant direct linear relationship (r = 0.397; P < 0.001) between serum PON3 and β-2-microglobulin concentrations (Fig. 3A). There were no significant differences in serum PON3 concentrations between HIV-infected patients with or without lipoatrophy [5.5 (2.0-12.8) versus 5.1 (2.4-10.3) mg/l, respectively; P = 0.644]. We observed a significant inverse relationship between serum PON3 concentrations and the duration of the antiretroviral therapy in patients under the NNRTI-based scheme (r =−0.250; P = 0.035; Fig. 3B) but not in patients receiving a PI-based scheme (r =−0.059; P = 0.408). When patients were classified according to the presence (n = 137) or absence (n = 41) of subclinical atherosclerosis, no significant differences in serum PON3 concentrations were found [5.2 (2.5-11.2) versus 5.4 (2.5-11.1) mg/l, respectively; P = 0.959]. Furthermore, there was no significant association between serum PON3 concentration and the quantitative value of the IMT (r = 0.047; P = 0.544). A multiple linear regression analysis showed significant and independent relationships of serum PON3 concentrations with oxidized LDL and with the use of NNRTI as the backbone of the antiretroviral therapy (Table 3). These results were confirmed when the use of antiretroviral treatment was substituted by its duration [NNRTI: B =−0.186 (0.080), β=−0.249, t =−2.083, P = 0.040; PI: B =−0.023 (0.050), β=−0.055, t =−0.419, P = 0.677].TABLE 3Multiple regression analysis of the determinants of serum PON3 concentration in HIV-infected patientsUnstandardized coefficientsStandardized coefficientsBStandard errorBetatPAge (years)−0.0.360.047−0.079−0.7530.454Gender (male)0.5280.7720.0760.6840.496BMI (kg/m)0.0980.1050.1020.9320.354Use of NNRTI−2.2760.875−0.329−2.6000.011Use of PI−0.8150.447−0.230−1.8210.072Lipoatrophy1.0350.8700.1441.1900.238Hepatitis C virus coinfection0.4860.7980.0720.6090.544HIV-1 viral load (<200 copies/ml)0.9320.8210.1361.1360.260CD4+ T-cell count (cells/mm)−0.0010.001−0.130−1.1700.246Total cholesterol (mmol/l)0.4570.3220.1791.4180.160HDL-cholesterol (mmol/l)−0.5290.831−0.074−0.6360.526Oxidized LDL (mmol/l)−0.0370.013−0.340−2.8330.006β-2-microglobulin (mg/l)0.1760.2920.1760.5500.550Dependent variable: PON3 (mg/l)BMI, body mass index; NNRTI, nonnucleoside analogs reverse transcriptase inhibitor; PI, protease inhibitor. Open table in a new tab BMI, body mass index; NNRTI, nonnucleoside analogs reverse transcriptase inhibitor; PI, protease inhibitor. Viral replication and some clinical manifestations of HIV infection involve a misbalance in reduction-oxidation (redox) status and free radical production (26.Schwarz K.B. Oxidative stress during viral infection: a review.Free Radic. Biol. Med. 1996; 21: 641-649Crossref PubMed Scopus (532) Google Scholar). Moreover, oxidative stress may be induced by antiretroviral treatments (27.Manda K.R. Banerjee A. Banks W.A. Ercal N. Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells.Free Radic. Biol. Med. 2011; 50: 801-810Crossref PubMed Scopus (67) Google Scholar). PON3 is an enzyme with lactonase activity (28.Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities.J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar), the physiological function of which is not completely understood, but evidence suggests that it has an antioxidant role by hydrolyzing oxidized lipid peroxides, similar to PON1 and PON2. Purified human and rabbit PON3 and recombinant PON3 have been shown to decrease macrophage oxidative stress and inhibit the in vitro oxidation of LDL (29.Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 30.Reddy S.T. Wadleigh D.J. Grijalva V. Ng C. Hama S. Gangopadhyay A. Shih D.M. Lusis A.J. Navab M. Fogelman A.M. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 542-547Crossref PubMed Scopus (303) Google Scholar, 31.Liu Y. Mackness B. Mackness M. Comparison of the ability of paraoxonases 1 and 3 to attenuate the in vitro oxidation of low-density lipoprotein and reduce macrophage oxidative stress.Free Radic. Biol. Med. 2008; 45: 743-748Crossref PubMed Scopus (37) Google Scholar, 32.Shih D.M. Xia Y.R. Yu J.M. Lusis A.J. Temporal and tissue-specific patterns of Pon3 expression in mouse: in situ hybridization analysis.Adv. Exp. Med. Biol. 2010; 660: 73-87Crossref PubMed Scopus (24) Google Scholar). The present study revealed a remarkable increase (about three times) in serum PON3 concentration in HIV-infected patients that may be clinically relevant. ROC analysis showed an AUC very close to 1.0, demonstrating a high sensitivity and specificity of serum PON3 measurement in distinguishing between HIV-infected and noninfected subjects. Interestingly, oxidized LDL levels were not significantly increased, but a significant inverse relationship was observed between their serum levels and those of PON3. These data support the concept that PON3 plays a protective role against oxidative stress and increased lipid peroxidation in HIV infection. Whether this increase in circulating PON3 is related to a higher cellular expression is, at present, unknown. However, a recent study reported a similar increase (five times) in Pon3 mRNA expression in late gestation, a physiological state with high oxidative stress (33.Belteki G. Kempster S.L. Forhead A.J. Giussani D.A. Fowden A.L. Curley A. Charnock-Jones D.S. Smith G.C. Paraoxonase-3, a putative circulating antioxidant, is systematically up-regulated in late gestation in the fetal rat, sheep, and human.J. Clin. Endocrinol. Metab. 2010; 95: 3798-3805Crossref PubMed Scopus (15) Google Scholar). Contrary to what we previously observed for PON1 in HIV-1 infection (5.Parra S. Alonso-Villaverde C. Coll B. Ferré N. Marsillach J. Aragonès G. Mackness M. Mackness B. Masana L. Joven J. et al.Serum paraoxonase-1 activity and concentration are influenced by human immunodeficiency virus infection.Atherosclerosis. 2007; 194: 175-181Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), serum PON3 concentrations were not significantly related with markers of lymphocyte recovery. We did not observe any association with CD4+ T-cell counts, and the correlation observed with β-2 microglobulin in the bivariate analysis disappeared in the multiple regression analysis. An interesting observation was the association of HCV coinfection with higher PON3 concentrations. These results confirm a recent report from our group describing increased serum PON3 concentrations in patients with chronic liver disease, mainly secondary to HCV infection (34.García-Heredia A. Marsillach J. Aragonès G. Guardiola M. Rull A. Beltrán-Debón R. Folch A. Mackness B. Mackness M. Pedro-Botet J. et al.Serum paraoxonase-3 concentration is associated with the severity of hepatic impairment in patients with chronic liver disease.Clin. Biochem. 2011; 44: 1320-1324Crossref PubMed Scopus (17) Google Scholar). In the present study, treatment of HIV-infected patients with NNRTI, but not with PI, was associated with a significant decrease of serum PON3 concentrations. The use of some nucleoside reverse transcriptase inhibitors (NRTI) and PIs is associated with an atherogenic lipoprotein profile (35.Tohyama J. Billheimer J.T. Fuki I.V. Rothblat G.H. Rader D.J. Millar J.S. Effects of nevirapine and efavirenz on HDL cholesterol levels and reverse cholesterol transport in mice.Atherosclerosis. 2009; 204: 418-423Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), but NNRTIs, such as efavirenz, promote antiatherogenic changes in HDL particles and function, including normalization of size and lipid composition, enhancement of reverse cholesterol transport, and improvement of antioxidant capacity (36.Pereira S.A. Batuca J.R. Caixas U. Branco T. Delgado-Alves J. Germano I. Lampreia F. Monteiro E.C. Effect of efavirenz on high-density lipoprotein antioxidant properties in HIV-infected patients.Br. J. Clin. Pharmacol. 2009; 68: 891-897Crossref PubMed Scopus (9) Google Scholar). A previous study from our group showed that NNRTI treatment was associated with a relative normalization of serum apoA-I and apoA-II, as well as HDL-cholesterol concentrations, in HIV-infected patients (37.Aragonès G. Beltrán-Debón R. Rull A. Rodríguez-Sanabria F. Fernández-Sender L. Camps J. Joven J. Alonso-Villaverde C. Human immunodeficiency virus-infection induces major changes in high-density lipoprotein particle size distribution and composition: the effect of antiretroviral treatment and disease severity.Clin. Chem. Lab. Med. 2010; 48: 1147-1152Crossref PubMed Scopus (11) Google Scholar), which is consistent with the reported beneficial effects of this compound on HDL composition and function. However, an alternative explanation is that the influence of NNRTI fades with time. Similar effects have been described for some antiretroviral drugs. Shlay et al. (38.Shlay J.C. Sharma S. Peng G. Gibert C.L. Grunfeld C. Long-term subcutaneous tissue changes among antiretroviral-naive persons initiating stavudine, zidovudine, or abacavir with lamivudine.J. Acquir. Immune Defic. Syndr. 2008; 48: 53-62Crossref PubMed Scopus (10) Google Scholar) reported that NRTI are associated with positive changes in subcutaneous tissue distribution during the early periods and negative changes in the late periods of treatment. We did not find any significant differences in genotype or haplotype of PON3 promoter gene polymorphisms between patients and controls, suggesting that genotype does not influence the course of the disease. In our previous report, we observed a moderate influence of some polymorphisms on serum PON3 concentration in the general population (18.Aragonès G. Guardiola M. Barreda M. Marsillach J. Beltran-Debon R. Rull A. Mackness B. Mackness M. Joven J. Simó J.M. et al.Measurement of serum paraoxonase-3 concentration: method evaluation, reference values and influence of genotypes in a population-based study.J. Lipid Res. 2011; 52: 1055-1061Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), but this is not the case in HIV-infected patients. Possibly, the upregulation of PON3 expression secondary to the infection masks the small effect of these polymorphisms. Unlike PON1 (6.Parra S. Marsillach J. Aragonès G. Beltrán R. Montero M. Coll B. Mackness B. Mackness M. Alonso-Villaverde C. Joven J. et al.Association of paraoxonase-1 gene haplotypes with the immunologic outcome of and metabolic disturbances and atherosclerosis in HIV-infected patients.J. Infect. Dis. 2010; 201: 627-634Crossref PubMed Scopus (23) Google Scholar), PON3 seems not to be associated with the presence of subclinical atherosclerosis in HIV-infected patients. Although lipid peroxidation and atherosclerosis are known to be strongly linked phenomena (39.Maskrey B.H. Megson I.L. Whitfield P.D. Rossi A.G. Mechanisms of resolution of inflammation: a focus on cardiovascular disease.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 1001-1006Crossref PubMed Scopus (127) Google Scholar), the pathophysiology of atherosclerosis is complex, and our results suggest that the protective effects of these enzymes differ under certain situations. Perhaps PON1 is more efficient in protecting against the alterations leading to atherosclerosis and PON3 is in some way involved in protection against infection. This is, to the best of our knowledge, the first in vivo evidence suggesting such a hypothesis, and it warrants further investigation. Another interesting point is the wider lipoprotein distribution of PON3 in HIV-infected patients. PON3 is observed in substantial amounts in the smallest HDL and in LDL particles. The accepted concept to-date is that PON1 and PON3 are exclusively transported in the bloodstream by HDL (12.Mackness B. Quarck R. Verreth W. Mackness M. Holvoet P. Human paraoxonase-1 overexpression inhibits atherosclerosis in a mouse model of metabolic syndrome.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1545-1550Crossref PubMed Scopus (145) Google Scholar) and are only associated with other lipoproteins in exceptional circumstances (40.Mackness M. Bouiller A. Hennuyer N. Mackness B. Hall M. Tailleux A. Duriez P. Delfly B. Durrington P. Fruchart J.C. et al.Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits.Biochem. Biophys. Res. Commun. 2000; 269: 232-236Crossref PubMed Scopus (77) Google Scholar). Perhaps the excess PON3 produced in HIV infection cannot be properly packed in the HDL particles, as the conformation of HDL-associated apoA-I leaves little free surface area for other proteins to bind (41.Huang R. Silva R.A. Jerome W.G. Kontush A. Chapman M.J. Curtiss L.K. Hodges T.J. Davidson W.S. Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma.Nat. Struct. Mol. Biol. 2011; 18: 416-422Crossref PubMed Scopus (181) Google Scholar). This may have resulted in some PON3 redistributing to LDL. The physiological implications of this observation require further investigation. In conclusion, this study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and that could be partially attenuated by treatment with some antiretroviral agents. Long-term, prospective studies are needed to further confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker. The authors thank Dr. John Teiber (University of Texas, Dallas, TX) and Drs. Dan Tawfik, Olga Khersonsky, and Leonid Gaidukov (Weizmann Institute of Science, Rehovot, Israel) for the generous gifts of purified PON3 and the TBBL reagent, respectively. The authors are indebted to Dr. Blai Coll (Vascular Medicine Unit, Hospital Universitari de Sant Joan) for his help in IMT measurements. Download .doc (.32 MB) Help with doc files acquired immunodeficiency syndrome fast-performance liquid chromatography hepatitis C virus human immunodeficiency virus intima-media thickness nonnucleoside reverse transcriptase inhibitor nucleoside reverse transcriptase inhibitor protease inhibitor paraoxonase single nucleotide polymorphism 5-thiobutyl butyrolactone
Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed nontargeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (P < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet.
Objectives Sheep-dippers report an acute flu-like condition (dippers' flu: DF) but the cause and relation to chronic disability are unknown. Methods In a case-referent study previously reported, 175 sheep dippers with chronic disability and 234 referents, sheep dippers in good health, completed an interview with information on dipping, type of pesticide used and health for each year 1970–2000 and gave blood for typing of PON1 polymorphisms. Results Reports of DF were much higher (66.3% 116/175) in the chronically unwell than in those without chronic ill-health (18.0% 42/234: OR=8.99 95% CI 5.69–14.21). No significant relation was seen between reported exposures and DF in those with chronic illness, but risk was higher with concentrate handling in those without. An R allele at position 192 on PON1 related to reports of DF both in those with chronic illness (OR=2.04 95% CI 1.08–3.87) and in those who started dipping after 1969 and were not chronically unwell (OR=2.52 95%CI 1.00–6.37). Interaction between handling diazinon concentrate and PON1 (192R) increased the risk of DF. No precipitating factor was identified in a case-crossover analysis. In the group without chronic illness those with 192R developed DF earlier (risk ratio 2.49 95%CI 1.03-6.02). Conclusion ‘Dippers’ flu' and chronic ill-health attributed to dipping share a common polymorphism (192R). The interaction between handling diazinon concentrate and PON1 genotype supports the conclusion that organophosphates may cause DF. Sheep dippers who are still healthy but experience ‘dippers’ flu' may be wise to further limit exposures to organophosphates.
Atherosclerosis in symptomatic peripheral arterial disease affects wide portions of numerous arteries in lower extremities. The resulting active inflammation in a considerable amount of arterial tissue facilitates systemic detection via measurement of inflammation-related variables. We reasoned that the combined assessment of defense against oxidative stress, in the form of paraoxonase-1 (PON1), and monocyte migration measured as circulating (C-C motif) ligand 2 (CCL2), may play a role in the evaluation of these patients. Plasma CCL2 and serum PON1-related variables, assessed by their interaction with functional genetic variants, were measured in a cross-sectional study in patients with symptomatic PAD. We found that PON1 activity and concentration were significantly lower and CCL2 concentration higher in PAD patients compared to controls, that the combination of plasma CCL2 and PON1- related values, especially PON1 concentration differentiated, almost perfectly, controls from patients and that the expression of CCL2 and PON1 generally co-localized in the atherosclerotic lesion. Since no association with genetic variants was found, such a relationship is probably the result of the disease. Our data suggest a coordinated role between CCL2 and PON1 that may be detected in blood with simple measurements and may represent an indicator of the extent of atherosclerosis. Keywords: Atherosclerosis, CCL2, inflammation, lactonase, monocyte chemoattractant protein-1, paraoxonase-1, peripheral vascular disease, polymorphism, peripheral arterial disease, Plasma, serum, disease, Cytokines, biomarkers, oxidative stress
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