Modulating reconstituted high density lipoprotein functionality to target the Pseudomonas aeruginosa quorum sensing system.

AIMS: The synthetic counterparts of serum high density lipoproteins (HDL; reconstituted HDL, reHDL) are assuming increasing importance as a therapeutic vector. They circulate not only in blood, but also outside the vascular compartment giving access to all body tissues. Presently, the therapeutic use of reHDL exploits inherent HDL functions. Our aim was to determine if HDL functionality could be modulated by attaching peptides not normally associated with the complex. MAIN METHODS: A peptide chimera was designed by linking the signal peptide of the HDL-associated enzyme paraoxonase-1 (PON1) to the coding region for the intracellular enzyme paraoxonase-2 (PON2). KEY FINDINGS: The signal peptide modified the properties of PON2, promoting its secretion from cells and binding to HDL. Enzyme activity of the chimera protein was highly stable. Conditioned HDL showed the functions of PON2 in its ability to hydrolyse typical PON2 substrates, namely homoserine lactones. Further in vitro studies showed that conditioned HDL was able to reduce the virulence of Pseudomonas aeruginosa. Both biofilm formation and the activation of the quorum sensing systems las and rhl, responsible for bacterial virulence, were significantly reduced. SIGNIFICANCE: The study provides proof of principal that the signal peptide of PON1 can be used to attach peptides to HDL and thus modulate HDL function. They may provide a vector that is ubiquitously distributed in extracellular body fluids for designing therapeutic strategies to address different pathophysiological states.

HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress.

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant capacity of the lipoprotein. In previous studies, we proposed that HDL promoted PON1 secretion by transfer of the enzyme from its plasma membrane location to HDL transiently anchored to the hepatocyte. This study examined whether PON1 can be transferred into cell membranes and retain its enzymatic activities and functions. Using Chinese hamster ovary and human endothelial cells, we found that recombinant PON1 as well as PON1 associated with purified human HDL was freely exchanged between the external medium and the cell membranes. Transferred PON1 was located in the external face of the plasma membrane of the cells in an enzymatically active form. The transfer of PON1 led to a gain of function by the target cells, as revealed by significantly reduced susceptibility to oxidative stress and significantly increased ability to neutralize the bacterial virulence agent 3-oxo-C(12)-homoserine lactone. The data demonstrate that PON1 is not a fixed component of HDL and suggest that the enzyme could also exert its protective functions outside the lipoprotein environment. The observations may be of relevance to tissues exposed to oxidative stress and/or bacterial infection.

The scavenger receptor class B, type I is a primary determinant of paraoxonase-1 association with high-density lipoproteins.

OBJECTIVE: To examine the contribution of the scavenger receptor (SR) BI to the mechanism by which high-density lipoprotein (HDL) acquires paraoxonase-1 (PON1). METHODS AND RESULTS: Serum PON1 activity contributes to the antioxidant capacity of HDLs and is suggested to be an independent risk factor for atherosclerosis. The association of PON1 with HDL is a major determinant of its serum activity levels. PON1 secretion was studied in stably transfected Chinese hamster ovary and HepG2 models. Complementary analyses were performed in transgenic models. Modulation of SR-BI expression, by SR-BI small and interfering RNA knockdown and pharmacologically, correlated with significant changes (P<0.01) in PON1 secretion to HDLs and very-low-density lipoproteins. Block lipid transport-1 (BLT1), which increases the affinity of HDL for SR-BI without modulating its expression, was associated with significant increases in secretion. Downregulating postsynaptic density 95/disc-large/zona occludens kinase in HepG2 reduced cell SR-BI protein and lowered enzyme secretion. Serum PON1 activity was significantly reduced in postsynaptic density 95/disc-large/zona occludens kinase knockout mice. CONCLUSIONS: The present study identifies SR-BI as a major determinant of the capacity of HDL to acquire PON1. It reinforces the concept of the receptor as a docking molecule, allowing communication between HDL and the cell, and extends the importance of SR-BI to HDL metabolism and function.

Expression of the hepatic Niemann-Pick C1 like 1 protein gene is sensitive to rosuvastatin treatment of primary human hepatocytes.

Statins act by reducing hepatic cholesterol synthesis, thus stimulating uptake of serum cholesterol. Statin therapy modulates a number of genes involved in hepatic cholesterol homeostasis. These have rarely been analyzed simultaneously in the same experimental setting, with virtually no studies of primary human hepatocytes. This study analyzed the efficacy of rosuvastatin in the coordinated regulation of a number of genes implicated in cholesterol metabolism in primary human hepatocytes. Expression of five cholesterol-related genes were significantly upregulated, notably the Niemann-Pick C1 like 1 protein, for whom functional studies have been essentially limited to the intestine. Two genes were significantly downregulated, including sterol recognition element binding protein-1 gene that is implicated in control of hepatic lipogenesis. The results show the coordinated regulation of several genes implicated in hepatic cholesterol homeostasis and suggest therapeutic targets that could complement that clinical action of statins.

The contribution of high density lipoprotein apolipoproteins and derivatives to serum paraoxonase-1 activity and function.

High density lipoproteins (HDL) not only provide a serum transport vector for paraoxonase-1 (PON1) but also contribute to enzyme activity, stability and, consequently, function. The contribution of the apolipoprotein (apo) components of HDL to overall PON1 activity and function is not clearly established. ApoAI appears of major importance in defining serum PON1 activity and stability, but in the context of an interaction with the phospholipid fraction of HDL. This may involve a role in establishing the architecture of the HDL particle that optimally integrates the PON1 peptide. As the second, major structural peptide of HDL, apoAII may accomplish a similar role. These apolipoproteins, together with others associated with HDL, may also exert a more indirect influence on PON1 function by sequestering oxidised lipids that could compromise enzyme activity. The latter has been exploited therapeutically to give rise to apolipoprotein mimetic peptides that may be useful in limiting oxidative stress within the lipoprotein system, thus permitting PON1 activity to be maximally expressed.

Effect of atorvastatin on paraoxonase1 (PON1) and oxidative status.

It has been proposed that paraoxonase1 (PON1), a high density lipoprotein (HDL)-associated esterase/lactonase, has anti-atherosclerotic properties. The activity of PON1 is influenced by PON1 polymorphisms. However, the influence of PON1 polymorphisms on PON1 activity and oxidative stress in response to lipid-lowering drugs remains poorly understood. The objective of the present study was to investigate the effects of atorvastatin on PON1 activity and oxidative status. The influence of PON1 polymorphisms on PON1 activity and oxidative status in response to atorvastatin treatment was also evaluated. In total, 22 hypercholesterolemic patients were treated with atorvastatin at a dose of 10 mg/day for 3 months. Lipid profile, lipid oxidation markers (malondialdehyde (MDA), conjugated diene (CD), total peroxides (TP)), total antioxidant substance (TAS), oxidative stress index (OSI), and paraoxonase1 activity were determined before and after treatment. L55M, Q192R, and T(-107)C PON1 polymorphisms were also determined. Atorvastatin treatment significantly reduced the levels of total cholesterol (24.5%), low density lipoprotein (LDL) cholesterol (25.4%), triglycerides (24.4%), CD (4.4%), MDA (15.2%), TP (13.0%) and OSI (24.0%), and significantly increased the levels of TAS (27.3%), and PON1 activity (14.0%). Interestingly, the increase in PON1 activity and the reduction in oxidative stress in response to atorvastatin were influenced only by the PON1 T-107C polymorphism. Atorvastatin treatment improved the lipid profile, lipid oxidation, and oxidative/antioxidative status markers including the activity of PON1 towards paraoxon. These beneficial effects may be attributed to the antioxidant properties of statins and the increase in PON1 activity. The increase in PON1 activity was enhanced by the PON1 T-107C polymorphism.

HDL subfraction distribution of paraoxonase-1 and its relevance to enzyme activity and resistance to oxidative stress.

The subfraction distribution of HDL-associated peptides has implications for their functions and the impact of pathological modifications to lipoprotein metabolism on these functions. We have analyzed the subfraction distribution of paraoxonase-1 (PON1) and the consequences for enzyme activity and stability. HDL subfractions were defined by the presence (LpA-I,A-II) or absence (LpA-I) of apolipoprotein A-II (apoA-II). PON1 was present in both subfractions, although increased concentrations of HDL were associated with significantly increased PON1 in LpA-I. ApoA-II did not modify the capacity of native human HDL or reconstituted HDL to promote PON1 secretion from cells or to stabilize enzyme activity, nor did apoA-II decrease PON1 activity when added to rabbit serum normally devoid of the apolipoprotein. LpA-I,A-II particles isolated from human serum or reconstituted HDL (LpA-I,A-II) showed a significantly greater capacity than HDL(LpA-I) to stabilize secreted PON1 and purified recombinant PON1 added to such particles. PON1 associated with apoA-II-containing particles showed greater resistance to inactivation arising from oxidation. ApoA-I, apoA-II, and LpA-I,A-II, but not LpA-I, were independent determinants of serum PON1 concentration and activity in multivariate analyses. PON1 is at least equally distributed between LpA-I and LpA-II,A-II HDL particles. This dichotomous distribution has implications for PON1 activity and stability that may impact on the physiological role of the enzyme.

Value-added education and smoking uptake in schools: a cohort study.

AIM: To show that schools achieving higher examination pass and lower truancy rates than expected, given that their pupil populations (high value-added schools) are associated with a lower incidence of smoking among pupils (13-14 years). DESIGN: Value-added scores for schools were derived from standardized residuals of two regression equations predicting separately the proportion of pupils passing high school diplomas and the half-days lost to truancy from the socio-economic and ethnic profiles of pupils. The risk of regular smoking at 1- and 2-year follow-up was examined in relation to the value-added score in a cohort of 8352 UK pupils. Random-effects logistic regression was used to adjust for baseline smoking status and other adolescent smoking risk factors. SETTING: A total of 52 schools, West Midlands, UK. PARTICIPANTS: Year 9 pupils aged 13-14 years (n = 8352) were followed-up after 1 year (n = 7444; 89.1% of original cohort) and 2 years (n = 6819; 84.6% of original cohort excluding pupils from two schools that dropped out). MEASUREMENTS: Regular smoking (at least one cigarette per week). FINDINGS: Schools with high value-added scores occurred throughout the socio-demographic spectrum. The odds ratio (95% confidence interval) for regular smoking for a 1 standard deviation increase in the value-added measure was 0.85 (0.73-0.99) at 1-year and 0.80 (0.71-0.91) at 2-year follow-ups. Baseline smoking status did not moderate this. CONCLUSIONS: Schools with high value-added scores are associated with lower incidence of smoking. Some schools appear to break the strong link between deprivation and smoking. Understanding the mechanisms could be of great public health significance.

Pharmacogenetic interaction between paraoxonase-1 gene promoter polymorphism C-107T and statin.

OBJECTIVE: The aims of this study were to compare the impact of transcription factors, together with statin, on the paraoxonase promoter alleles defined by the C(-107T) polymorphism and to more clearly define regions of the paraoxonase promoter implicated in the actions of transcription factors. METHODS: Expression studies of promoter alleles were performed with reporter gene cassettes transfected into HepG2 cells, complemented by nuclease protection assays, electrophoretic mobility shift assays and statin therapy in patients. RESULTS: One region only of the minimal promoter fragment that up-regulates activity was protected by transcription factors and nuclear extracts. It spanned nucleotides -119 to -100, encompassing the C(-107)T polymorphism. Sp1 was effective alone in protecting this region, with its effect greatly enhanced by SREBP-2. The protective effect was much stronger for the C vs. T promoter allele. Expression studies confirmed the stimulatory influence of SREBP-2 was significantly stronger for the C promoter. Simvastatin up-regulated promoter activity of the C allele, but had a minor effect on the T allele. Hypercholesterolemic patients homozygous for the C allele showed a significant increase in serum paraoxonase-1 activity and mass during treatment with simvastatin, whereas patients homozygous for the T allele showed no increase. CONCLUSIONS: The study has delimited the region responsive to transcription factors to a sequence surrounding the C(-107)T polymorphism of the paraoxonase-1 gene, and demonstrated an interaction at this sequence between Sp1 and SREBP-2. SREBP-2 and statin strongly up-regulated the C, but not the T allele. The results suggest a pharmacogenetic interaction between the promoter and simvastatin, which can influence serum paraoxonase in patients.

HDL oxidation compromises its influence on paraoxonase-1 secretion and its capacity to modulate enzyme activity.

OBJECTIVE: The purpose of this study was to analyze the consequences of HDL oxidation for paraoxonase-1 metabolism and function. METHODS AND RESULTS: HDL was oxidized with AAPH, copper ions, and hypochlorite. Secretion studies were performed using human paraoxonase-1-transfected cells lines and primary rat hepatocytes. Stability studies were performed with recombinant paraoxonase. Conditioned medium had significantly reduced paraoxonase-1 when Cu or AAPH-oxidized HDL was the acceptor complex (P<0.01); reduction was dose-dependent on the degree of oxidation. Oxidized HDL had a reduced capacity to stabilize/improve activity of secreted paraoxonase-1. Reduced secretion could not be attributed to enzyme inactivation by lipoperoxides, reduced binding affinity of HDL, or oxidation of the lipid component alone. Hypochlorite oxidation of HDL did not modify HDL-mediated paraoxonase-1 release, but activity of HDL-associated paraoxonase-1 was particularly sensitive to such treatment. CONCLUSIONS: AAPH and copper, but not hypochlorite, oxidation of HDL compromises its ability to promote release of paraoxonase-1 and stabilize enzyme activity. HDL-associated paraoxonase-1 is highly sensitive to hypochlorite. Reducing paraoxonase-1 renders HDL susceptible to oxidation, which may compromise HDL function. It provides a novel example at the HDL level of the detrimental effects of oxidative stress, and underlines the need for further evaluation of the consequences of HDL oxidation.

Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.

Paraoxonase-1 (PON1) requires a suitable acceptor complex for its secretion from producing cells. The serum lipoprotein, high-density lipoprotein (HDL) has been shown to accomplish this function, whereas low-density lipoproteins are ineffective. The present study examined the influence of the third serum lipoprotein subclass, very low density lipoproteins (VLDL), on PON1 secretion. VLDL were shown to promote secretion of PON1 from a transfected Chinese hamster ovary model and from transfected hepatocytes in a high-affinity, saturable manner. The effects of HDL and VLDL were not additive, suggesting that they may employ a common secretion pathway. VLDL was able to stabilise secreted PON1 enzyme activity, but less effectively than stabilisation by HDL. Following co-incubation of VLDL and HDL, the majority of PON1 accumulated in HDL even if HDL was added after initial association of the enzyme with VLDL. VLDL to HDL transfer of PON1 was rapid and did not require lipolysis of VLDL. Low levels of active PON1 were associated with VLDL in human serum, and VLDL-associated enzyme activity was proportional to serum triglyceride concentrations. Serum triglycerides were positively associated with whole serum PON1 mass but negatively associated with specific activity. PON1-enriched VLDL was more resistant to oxidation in vitro. The present study suggests that the triglyceride transport vector, VLDL, can modulate PON1 metabolism and activity. This is due, in part, to an influence of the lipoprotein on PON1 secretion. PON1 was associated with VLDL in human serum, where triglycerides correlated independently with variations in serum mass and activity of the enzyme. VLDL-associated PON1 exerted an anti-oxidative effect, which may be of physiological benefit.

The importance of high-density lipoproteins for paraoxonase-1 secretion, stability, and activity.

The association of paraoxonase-1 (PON1) with high-density lipoproteins (HDL) is a prerequisite for maintaining normal serum activity of the enzyme. The lipoprotein furnishes an amphipathic environment to shield the hydrophobic, N-terminal region of the enzyme, and such an environment may also be necessary for interaction of PON1 with its substrates. HDL provides the optimal physiological acceptor complex, in terms of both stimulating PON1 secretion and stabilizing the secreted peptide. Lipid and peptide components of HDL contribute to these effects, such that modulating HDL composition influences PON1 activity and function. In this context, understanding how PON1 associates with HDL, what governs the association, and the mechanism by which the PON1-HDL complex exerts its antioxidant function is of particular physiological relevance. Moreover, HDL is subject to substantial compositional variations under both normal and pathological metabolic conditions. It has implications for the influence of the enzyme on cardiovascular risk, as normal enzyme activity may not correlate with optimal functional (antioxidant) efficiency. We review evidence that HDL lipid and protein components interact to promote PON1 secretion and maintain serum enzyme activity. Emerging data on how the enzyme associates with HDL are discussed, and the consequences for PON1 function of modifications to HDL are outlined. Finally, we highlight questions concerning the HDL-PON1 association that remain unanswered but are of particular importance in defining PON1 efficiency.

Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.

PON1 (paraoxonase-1) is an HDL (high-density lipoprotein)-associated enzyme capable of hydrolysing diverse substrates from OP (organophosphate) toxins to oxidized phospholipids. As such, it has been linked with both the prevention of OP poisoning and inhibition of atherosclerosis initiated by oxidatively modified LDL (low-density lipoprotein). Mice deficient in PON1 are more susceptible to OP poisoning and oxidative stress and more prone to develop atherosclerosis than their wild-type siblings. There are a number of polymorphisms in the PON1 gene which affect serum PON1 activity and concentration. Many (but not all) studies in human populations have suggested that these polymorphisms may be a risk factor for atherosclerosis. The serum concentration of PON1 across the general population is highly variable and there is some debate as to whether genotype or phenotype (i.e. the quantity or quality of the enzyme) is most accurately associated with risk of disease development. What is clear is that factors influencing serum levels of PON1, be they genetic or environmental, will, in turn, affect the capacity of HDL to protect LDL from oxidation and, consequently, may be linked to atherosclerosis. This review will focus on mechanisms which determine the serum concentration of PON1, including gene expression and genetic polymorphisms, protein secretion and association with HDL, pharmacological and environmental factors.

Simvastatin modulates expression of the PON1 gene and increases serum paraoxonase: a role for sterol regulatory element-binding protein-2.

BACKGROUND: The HDL-associated enzyme paraoxonase protects LDLs from oxidative stress. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) appear to favorably influence the atherosclerotic process by different mechanisms. The present study examined the influence of simvastatin on paraoxonase expression and serum paraoxonase levels. METHODS AND RESULTS: Simvastatin upregulated in a dose-dependent manner the activity of the promoter of the paraoxonase gene in expression cassettes transfected into HepG2 cells. Upregulation could be blocked by mevalonate and other intermediates of the cholesterol biosynthetic pathway. Simvastatin increased nuclear factors, notably sterol regulatory element-binding protein-2, capable of binding to the paraoxonase promoter; this was also blocked by mevalonate. Sterol regulatory element-binding protein-2 upregulated promoter activity in vitro. Patients treated with statin showed a significant increase in serum concentrations and activities of paraoxonase. CONCLUSIONS: The data indicate that simvastatin can modulate expression in vitro of the antioxidant enzyme paraoxonase and is associated with increased serum paraoxonase concentration and activity. It is consistent with effects of simvastatin treatment, which have the potential to influence beneficially antiatherogenic mechanisms at the HDL level. The study provides evidence for 1 molecular mechanism by which paraoxonase gene expression could be regulated.

Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position - 107, implicating the Sp1 transcription factor.

Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P <0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (-107, -824 and -907), the -107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the -107 site (-107C) alone. No significant contribution was observed for the -824 and -907 sites. The coding-region Leu(55)-->Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the -107 site. A molecular basis for the effect of the -107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (-107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(-107)T polymorphism for gene expression in vivo. Differences in the affinity of the -107C and -107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the -107 polymorphism.

Paraoxonase-1 L55M polymorphism is associated with an abnormal oral glucose tolerance test and differentiates high risk coronary disease families.

Polymorphisms of the gene for the antioxidant enzyme, paraoxonase-1 (PON1), have been identified as risk factors for coronary disease (CHD), notably in diabetic patients. The polymorphisms have also been linked with other diabetic complications. The present study analyzed glucose metabolism as a function of PON1 polymorphisms in young healthy nondiabetic men from families with premature CHD and matched controls. The L55M PON1 polymorphism was independently associated with the glucose response to an oral glucose tolerance test. LL homozygotes had significantly impaired glucose disposal (P = 0.0007) compared with (LM+MM) genotypes. It was particularly marked for subjects from high CHD risk families and differentiated them from matched controls (P = 0.049). The area under the glucose curve (P = 0.0036) and the time to peak glucose value (P = 0.026) were significantly higher in the LL carriers, whereas the insulin response was slower (P = 0.013). Insulin resistance did not differ between L55M genotypes. There was a trend for reduced pancreatic beta-cell function as measured by glucose-induced insulin secretion (LL vs. LM vs. MM, 20.26 vs. 23.74 vs. 25.60; P = 0.077). The frequency of the L55 allele decreased significantly (P = 0.028) across regions defining a north-south European axis. No significant differences for the glucose response or case-control populations were observed as a function of the PON1 Q192R polymorphism. The study demonstrates an association between PON1 gene polymorphisms and glucose metabolism. The L55M-glucose interaction differentiated offspring of high CHD risk families, suggesting that it may be of particular relevance for vascular disease and possibly other diabetic complications.

Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism.

Paraoxonase-1 (PON1) is a high density lipoprotein (HDL)-associated serum enzyme that protects low density lipoproteins from oxidative modifications. There is a relative lack of information on mechanisms implicated in PON1 release from cells. The present study focused on a model derived from stable transfection of CHO cells, to avoid co-secretion of apolipoprotein (apo) A-I and lipids, which could lead to formation of HDL-like complexes. Our results indicate that, in the absence of an appropriate acceptor, little PON1 is released. The results designate HDL as the predominant, physiological acceptor, whose efficiency is influenced by size and composition. Neither lipid-poor apoA-I or apoA-II nor low density lipoproteins could substitute for HDL. Protein-free phospholipid complexes promoted PON1 release. However, the presence of both apolipoprotein and phospholipid were necessary to promote release and stabilize the enzyme. Immunofluorescence studies demonstrated that PON1 was inserted into the external membrane of CHO cells, where it was enzymatically active. Accumulation of PON1 in the cell membrane was not influenced by the ability of the cell to co-secrete of apoA-I. Release appeared to involve desorption by HDL; human and reconstituted HDL promoted PON1 release in a saturable, high affinity manner (apparent affinity 1.59 +/- 0.3 microg of HDL protein/ml). Studies with PON1-transfected hepatocytes (HuH-7) revealed comparable structural features with the peptide located in a punctate pattern at the external membrane and enzymatically active. We hypothesize that release of PON1 involves a docking process whereby HDL transiently associate with the cell membrane and remove the peptide from the external membrane. The secretory process may be of importance for assuring the correct lipoprotein destination of PON1 and thus its functional efficiency.