Marie-Claude Brulhart-Meynet

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.

Improving Reconstituted HDL Composition for Efficient Post-Ischemic Reduction of Ischemia Reperfusion Injury

Background New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. Objective The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. Methods and Results The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. Conclusion HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

Myeloperoxidase and paraoxonase-1 in type 2 diabetic patients *

BACKGROUND AND AIMS Reduced high density lipoproteins (HDL) and increased oxidative stress are features of type 2 diabetes. Myeloperoxidase is an oxidative enzyme partly associated with HDL and causing HDL dysfunction. It is an independent risk factor for cardiovascular disease. Paraoxonase-1 is an HDL-associated enzyme that protects against cardiovascular disease and is reduced in diabetes. The present study examined if serum myeloperoxidase was (i) increased in type 2 diabetes, (ii) correlated with paraoxonase-1 activity. METHODS AND RESULTS The study was based on cross-sectional analyses of serum myeloperoxidase and paraoxonase-1 in type 2 diabetic patients and non-diabetic participants, with and without cardiovascular disease. Serum myeloperoxidase concentrations were not increased in type 2 diabetic patients without cardiovascular disease compared to non-diabetic controls. They were significantly higher in type 2 patients and non-diabetic patients with angiographically confirmed coronary disease. HDL-associated myeloperoxidase was correlated with serum myeloperoxidase (r=0.80, p<0.001) but not HDL-cholesterol (r=0.08) or apolipoprotein AI (r=0.08). Multivariate analyses showed serum myeloperoxidase to be an independent determinant of paraoxonase activities (arylesterase, p=0.024; paraoxonase, p=0.026). CONCLUSIONS Myeloperoxidase is an independent, negative determinant of paraoxonase-1 activity, which may be one mechanism by which it promotes HDL dysfunction and increases cardiovascular risk. Increased serum myeloperoxidase is not a feature of type 2 diabetes in the absence of overt cardiovascular disease. The level of HDL-associated myeloperoxidase is determined by the serum concentration of the enzyme suggesting that, in the context of reduced HDL concentrations in diabetic patients, myeloperoxidase may have a greater impact on HDL function.

Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection

Objective—The dyslipidemia of type 2 diabetes mellitus has multiple etiologies and impairs lipoprotein functionality, thereby increasing risk for cardiovascular disease. High-density lipoproteins (HDLs) have several beneficial effects, notably protecting the heart from myocardial ischemia. We hypothesized that glycation of HDL could compromise this cardioprotective effect. Approach and Results—We used in vitro (cardiomyocytes) and ex vivo (whole heart) models subjected to oxidative stress together with HDL isolated from diabetic patients and nondiabetic HDL glycated in vitro (methylglyoxal). Diabetic and in vitro glycated HDL were less effective (P<0.05) than control HDL in protecting from oxidative stress. Protection was significantly, inversely correlated with the degree of in vitro glycation (P<0.001) and the levels of hemoglobin A1c in diabetic patients (P<0.007). The ability to activate protective, intracellular survival pathways involving Akt, Stat3, and Erk1/2 was significantly reduced (P<0.05) using glycated HDL. Glycation reduced the sphingosine-1-phosphate (S1P) content of HDL, whereas the S1P concentrations of diabetic HDL were inversely correlated with hemoglobin A1c (P<0.005). The S1P contents of in vitro glycated and diabetic HDL were significantly, positively correlated (both <0.01) with cardiomyocyte survival during oxidative stress. Adding S1P to diabetic HDL increased its S1P content and restored its cardioprotective function. Conclusions—Our data demonstrate that glycation can reduce the S1P content of HDL, leading to increased cardiomyocyte cell death because of less effective activation of intracellular survival pathways. It has important implications for the functionality of HDL in diabetes mellitus because HDL-S1P has several beneficial effects on the vasculature.

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. Conclusion—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.

Paraoxonase-1 promoter polymorphism C--107T and serum apolipoprotein AI interact to modulate serum paraoxonase-1 status.

Objectives The objective was to examine the hypothesis that modifications to paraoxonase-1 specific activity (SP, activity per unit mass peptide) could contribute to serum paraoxonase-1 status, a determinant of the clinical efficacy of the enzyme. Methods Enzyme activities and concentrations were determined in a large population (n=912) of patients and controls. SP were subsequently examined as a function of paraoxonase-1 gene polymorphisms, plasma lipids and lipoproteins, and physiological and pathophysiological parameters. Results Pathophysiological parameters (diabetes, metabolic syndrome, smoking, aging) did not promote variations in paraoxonase-1 SP, whilst coronary disease lowered SP (P<0.003). No serum lipid, apolipoprotein or lipoprotein component had an impact on specific activity, with the exception of apolipoprotein AI (P<0.005, both substrates). The paraoxonase-1 promoter C−107T and Q192R polymorphisms influenced SP and, together with apolipoprotein AI, were highly significant, independent determinants in regression models. There was an interaction between apolipoprotein AI and the C−107T polymorphism, which significantly modulated SP and serum paraoxonase-1 status. Conclusions Enzyme inactivation giving rise to modulated activity per unit mass of peptide is not a major contributor to pathological effects of disease on serum paraoxonase-1 status. The C−107T polymorphism and serum apolipoprotein AI have major impacts individually on SP and also provide an example of gene-environment interaction to modulate such activities. These effects accentuate the differences between −107C and −107T allele carriers in terms of serum paraoxonase-1 status. The data underline the complexity of the factors that determine serum paraoxonase-1 status and suggest that the latter would benefit from therapeutic modulation of serum high density lipoproteins.

High-density lipoprotein-associated sphingosine-1-phosphate activity in heterozygous familial hypercholesterolaemia

Patients with heterozygous familial hypercholesterolaemia (FH) suffer from high plasma cholesterol and an environment of increased oxidative stress. We examined its potential effects on high‐density lipoprotein (HDL)‐associated sphingosine‐1‐phosphate (S1P) content (HDL‐S1P) and HDL‐mediated protection against oxidative stress, both with and without statin treatment.

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.

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism

Context Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology.