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Background— Gap junctions allow the direct exchange of ions and small molecules between cells in contact, thus coordinating physiological processes such as cell growth and differentiation. We have recently demonstrated increased expression of the gap junction protein connexin43 (Cx43) in specific subsets of cells in atherosclerotic lesions. Because the development of atherosclerosis depends critically on paracrine cell-to-cell interactions, we hypothesized that direct intercellular communication via gap junctions may be another factor controlling atherogenesis. Methods and Results— The role of Cx43 in atherogenesis was examined by use of both a genetic and a pharmacological approach. First, atherosclerosis-susceptible LDL receptor–deficient (LDLR −/− ) mice with normal (Cx43 +/+ ) or reduced (Cx43 +/− ) levels of Cx43 were fed a cholesterol-rich diet for 14 weeks. The progression of atherosclerosis was reduced by 50% ( P +/− LDLR −/− mice compared with Cx43 +/+ LDLR −/− controls. Atheroma in Cx43 +/− LDLR −/− mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells. Next, we observed that HMG-CoA reductase inhibitors, or “statins,” lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, reduced Cx43 expression in primary human vascular cells in vitro. Atheroma of LDLR −/− mice treated orally with pravastatin contained fewer inflammatory cells and exhibited thicker fibrous caps than controls. This was associated with reduced Cx43 expression in lesions of statin-treated mice. Conclusions— These data indicate a critical role for Cx43-mediated gap junctional communication in atherosclerotic plaque formation.

During the last decades, the improvements in the assessment and management of cardiovascular risk have been successful in the reduction of acute cardiovascular outcomes. Among different prevention and therapeutic strategies, treatments targeting HDL increase have been indicated as promising interventions from both basic research and clinical studies. Although this therapeutic approach still requires confirmatory findings from large clinical trials, emerging evidence indicates that it would be particularly useful in some patient categories characterized by accelerated atherosclerotic diseases. In this regard, HIV-infected patients (under antiretroviral treatment or not) have been recently indicated as an appropriate target population. The present review will discuss the promising role of HDL in HIV-related cardiovascular pathophysiology and update novelties from clinical studies targeting HDL increase in HIV patients.

In vitro and animal studies point to autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) as possible mediators of cardiovascular (CV) disease involving several mechanisms such as basal heart rate interference mediated by a mineralocorticoid receptor–dependent L-type calcium channel activation, and a direct pro-inflammatory effect through the engagement of the toll-like receptor (TLR) 2/CD14 complex. Nevertheless, the possible implication of these receptors in the pro-arrhythmogenic effect of anti-apoA-1 antibodies remains elusive. We aimed at determining whether CD14 and TLRs could mediate the anti-apoA-1 IgG chronotropic response in neonatal rat ventricular cardiomyocytes (NRVC). Blocking CD14 suppressed anti-apoA-1 IgG binding to NRVC and the related positive chronotropic response. Anti-apoA-1 IgG alone induced the formation of a TLR2/TLR4/CD14 complex, followed by the phosphorylation of Src, whereas aldosterone alone promoted the phosphorylation of Akt by phosphatidylinositol 3-kinase (PI3K), without affecting the chronotropic response. In the presence of both aldosterone and anti-apoA-1 IgG, the localization of TLR2/TLR4/CD14 was increased in membrane lipid rafts, followed by PI3K and Src activation, leading to an L-type calcium channel–dependent positive chronotropic response. Pharmacological inhibition of the Src pathway led to the decrease of L-type calcium channel activity and abrogated the NRVC chronotropic response. Activation of CD14 seems to be a key regulator of the mineralocorticoid receptor–dependent anti-apoA-1 IgG positive chronotropic effect on NRVCs, involving relocation of the CD14/TLR2/TLR4 complex into lipid rafts followed by PI3K and Src-dependent L-type calcium channel activation.

High-density lipoproteins (HDL) are complexes of multiple talents, some of which have only recently been recognised but all of which are under active investigation. Clinical interest initially arose from their amply demonstrated role in atherosclerotic disease with their consequent designation as a major cardiovascular disease (CVD) risk factor. However, interest is no longer confined to vascular tissues, with the reports of impacts of the lipoprotein on pancreatic, renal and nervous tissues, amongst other possible targets. The ever-widening scope of HDL talents also encompasses environmental hazards, including infectious agents and environmental toxins. In almost all cases, HDL would appear to have a beneficial impact on health. It raises the intriguing question of whether these various talents emanate from a basic ancestral function to protect the cell.The following chapter will illustrate and review our current understanding of some of the functions attributed to HDL. The first section will look at the antioxidative functions of HDL and possible mechanisms that are involved. The second section will focus specifically on paraoxonase-1 (PON1), which appears to bridge the divide between the two HDL functions discussed herein. This will lead into the final section dealing with HDL as a detoxifying agent protecting against exposure to environmental pathogens and other toxins.

High-density lipoproteins (HDL) are complexes of multiple talents, some of which have only recently been recognised but all of which are under active investigation. Clinical interest initially arose from their amply demonstrated role in atherosclerotic disease with their consequent designation as a major cardiovascular disease (CVD) risk factor. However, interest is no longer confined to vascular tissues, with the reports of impacts of the lipoprotein on pancreatic, renal and nervous tissues, amongst other possible targets. The ever-widening scope of HDL talents also encompasses environmental hazards, including infectious agents and environmental toxins. In almost all cases, HDL would appear to have a beneficial impact on health. It raises the intriguing question of whether these various talents emanate from a basic ancestral function to protect the cell.The following chapter will illustrate and review our current understanding of some of the functions attributed to HDL. The first section will look at the antioxidative functions of HDL and possible mechanisms that are involved. The second section will focus specifically on paraoxonase-1 (PON1), which appears to bridge the divide between the two HDL functions discussed herein. This will lead into the final section dealing with HDL as a detoxifying agent protecting against exposure to environmental pathogens and other toxins.

Die neuen amerikanischen Guidelines des American College of Cardio­logy und der American Heart Association empfehlen, die Schwelle zur Verschreibung von Statinen in der kardiovaskularen Primarpravention zu senken und auf die Zielwerte fur LDL-Cholesterin zu verzichten, um am haufigsten stark wirksame Statine einzusetzen. Die Schweizer Arbeitsgruppe Lipide und Atherosklerose (AGLA) glaubt, dass diese Empfehlungen in der Schweiz nicht umgesetzt werden sollten.

Oxidative stress could contribute to diabetes and its complications by predisposing to insulin resistance. Lipid peroxidation products are thought to be one mechanism involved in reduced insulin sensitivity. The serum enzyme, paraoxonase-1, protects lipoprotein lipids from oxidation. We examined the hypothesis that paraoxonase-1 could be associated with abnormal serum glucose concentrations in non-diabetic patients.Serum paraoxonase-1 activities and concentrations, as well as paraoxonase-1 gene polymorphisms, were analysed as a function of fasting glucose concentrations in non-diabetic patients and in Type II (non-insulin-dependent) diabetic patients.Serum paraoxonase-1 activities and concentrations were lower (p0.05) in non-diabetic patients with abnormal fasting glucose concentrations. It was due to a higher frequency of low expressor paraoxonase-1 promoter genotypes in patients with abnormal glucose control. Promoter polymorphisms were independent determinants of abnormal fasting glucose concentrations. Low expressor genotypes were associated with higher glucose concentrations in non-diabetic patients (p = 0.046) and a trend to higher concentrations in Type II diabetic patients. The coding region paraoxonase-1 polymorphisms L55 M and Q192R was not associated with differences in fasting glucose. CONCLUSION/INTERPRETATION. The promoter polymorphism C(-107)T is a marker for abnormal fasting glucose concentrations in non-diabetic patients. It could indicate an active role for paraoxonase-1, possibly pre-disposing to insulin resistance, or linkage of paraoxonase-1 polymorphisms with other gene products implicated in glucose metabolism.

Low density lipoproteins (LDLs) play an important role in the pathogenesis of atherosclerosis. LDL has been shown to be mitogenic and proapoptotic for vascular smooth muscle cells. However, the mechanisms are poorly understood and may result from an alteration in intracellular mitogenic signaling either directly by LDL or indirectly through an autocrine effect involving growth factor secretion and/or growth factor receptor expression. Insulin-like growth factor-1 (IGF-1) is an autocrine/paracrine factor for vascular smooth muscle cells and has potent anti-apoptotic effects. Thus, we hypothesized that part of the proliferative responses to LDLs may be explained by its modulation of IGF-1 or IGF-1 receptor (IGF-1R) expression. Treatment of rat vascular smooth muscle cells with increasing doses of native LDL dose-dependently increased IGF-1 mRNA by up to 2.6-fold; however, native LDL had no effect on IGF-1R mRNA expression. In contrast, the same doses of oxidized LDL significantly reduced IGF-1 and IGF-1R mRNA by 80 and 61%, respectively, and reduced IGF-1 and IGF-1R protein expression by 63 and 46%. In addition, native and oxidized LDL significantly increased IGF-1-binding protein-2 and IGF-1-binding protein-4 expression as measured by Western ligand blot. Most interestingly, anti-IGF-1 antiserum completely inhibited LDL-induced but not serum-induced increase in 3H-thymidine incorporation, indicating a requirement for IGF-1 in the LDL-stimulated mitogenic signaling pathway. In summary, these results suggest that native and oxidized LDLs have differential effects on IGF-1 and IGF-1R expression. Because IGF-1 is a potent survival factor for vascular smooth muscle cells, our findings suggest that moderately oxidized LDL may favor proliferation of smooth muscle cells, whereas oxidized LDL may contribute to plaque apoptosis by local depletion of IGF-1 and IGF-1R.

Background: The influence of hyperinsulinemia on concentrations of lipoprotein subfractions in obese, nondiabetic persons has not been clarified. Objective: We analyzed VLDL and LDL subfractions before and after a euglycemic, hyperinsulinemic clamp. Design: Lipoprotein subfractions were isolated from plasma samples obtained in the basal state and after a 4-h clamp from obese patients, obese patients with type 2 diabetes, and nonobese control subjects. Results: Hyperinsulinemia tended to reduce concentrations (x: 20%) of large, triacylglycerol-rich VLDL, in obese patients but had a minor effect on VLDL 2 and VLDL 3 . Placing obese patients into insulin-sensitive and insulin-resistant subgroups revealed distinct effects of the degree of insulin sensitivity on VLDL. VLDL, concentrations decreased by a mean of 38% (P < 0.05) in insulin-sensitive patients after the clamp, similar to but less marked than the decrease observed in control subjects (x: 62%; P < 0.01). VLDL 1 concentrations did not change significantly after the clamp in insulin-resistant patients (and patients with type 2 diabetes), whereas VLDL 3 concentrations decreased in both groups, in contrast with the changes seen in the insulin-sensitive patients and control subjects. Acute hyperinsulinemia modified the LDL subfraction profile toward a greater prevalence of small, dense LDLs in insulin-resistant patients and patients with type 2 diabetes. Conclusions: Insulin resistance appears to be the primary determinant of the modifications to VLDL subfraction concentrations. Our results suggest a continuum of impaired insulin action on VLDL, ranging from that in healthy persons to that in patients with type 2 diabetes, in which obese patients occupy a transition state. Insulin resistance may also play a role in detrimental modifications to the LDL profile by allowing the development of hypertriglyceridemia.

Paraoxonase is a high density lipoprotein (HDL) associated enzyme with a hypothesised role in the protection of low density lipoproteins (LDL) from oxidative stress. The present study examined paraoxonase in several genetically distinct HDL deficiency states. Despite reduction or even absence of detectable HDL, enzyme activity was present in sera from A-I-Pisa, A-I-Helsinki, A-I-Milano and Tangier patients. Both enzyme activities and peptide concentrations were modulated (reduced) but specific activities were broadly similar to controls, suggesting an impact on peptide concentration rather than an inhibition of enzyme activity. Despite the absence of HDL in A-I-Pisa and Tangier subjects, there was no association of paraoxonase with very low density lipoproteins or LDL. Paraoxonase function is maintained in HDL deficient states. It implies that certain HDL-associated anti-atherogenic processes may not be entirely compromised by HDL deficiency. This has important implications for the cardiovascular risk associated with modulated HDL concentrations.

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.

High density lipoprotein (HDL) cholesterol has beneficial effects beyond its atheroprotective function in reverse cholesterol transport, including cardioprotection against ischemia reperfusion (IR) injuries. Two major constituents of HDL, namely the structural protein apolipoprotein AI (apoAI) and the sphingolipid sphingosine-1-phosphate (S1P) appear to contribute to this cardioprotective effect via the activation of intrinsic prosurvival signaling pathways that still remain to be clarified. Recently, a powerful prosurvival signaling pathway, termed the survivor activating factor enhancement (SAFE) pathway, which involves the activation of signal transducer and activator of transcription 3 (STAT3) and tumor necrosis factor α (TNF), has been shown to protect against ischemia-reperfusion injuries. The present review summarizes the evidence for the roles of HDL and S1P in cardioprotection and discusses the signaling pathways that have been implicated. It thus provides support for our contention that S1P should be considered in potential formulations of reconstituted HDL (reHDL) that may be tested for cardioprotection against coronary artery disease via the activation of the SAFE pathway.

A method of extracting accurate Green's functions from ab initio plane wave pseudopotential calculations is presented. The method is tested by comparison with an independent source of real-space Green's functions on a finite difference grid. Some of the uses of the extracted Green's functions are outlined.

High density lipoproteins (HDL) protect against ischemia reperfusion injury (IRI). However the precise mechanisms are not clearly understood. The novel intrinsic prosurvival signaling pathway named survivor activating factor enhancement (SAFE) path involves the activation of tumor necrosis factor (TNF) alpha and signal transducer and activator of transcription 3 (STAT3). SAFE plays a crucial role in cardioprotection against IRI. We propose that HDL protect against IRI via activation of the SAFE pathway and modulation of the mitochondrial permeability transition pore (mPTP) opening.Isolated mouse hearts were subjected to global ischemia (35 min) followed by reperfusion (45 min). HDL were given during the first 7 min of reperfusion. In control hearts, the post-reperfusion infarct size was 41.3 ± 2.3%. Addition of HDL during reperfusion reduced the infarct size in a dose-dependent manner (HDL 200 μg protein/ml: 25.5 ± 1.6%, p0.001 vs. control). This protective effect was absent in TNF deficient mice (TNF-KO) or cardiomyocyte-STAT3 deficient mice (STAT3-KO). Similarly, HDL, given as a preconditioning stimulus, improved cell survival and inhibited mPTP opening in isolated cardiomyocytes subjected to simulated ischemia. These protective responses were inhibited in cardiomyocytes from TNF-KO and STAT3-KO mice.Our data demonstrate that HDL protect against IRI by inhibition of mPTP opening, an effect mediated via activation of the SAFE pathway.

OBJECTIVE To examine the prevalence of cardiovascular disease in diabetic patients as a function of apolipoprotein (apo) E polymorphism. RESEARCH DESIGN AND METHODS The apo E phenotypes and plasma lipid, lipoprotein, and apo levels were determined for 517 Italian diabetic patients. The prevalence of cardiovascular disease (defined as ischemic heart disease [IHD] and/or peripheral vascular disease and/or cerebrovascular disease) was assessed as a function of apo E polymorphism at entry and after 4 years. RESULTS The occurrence of vascular disease did not differ significantly between diabetic patients in the various categories of apo E phenotype either at entry into the study or after 4 years. When expressed as a percentage of patients with disease, we observed—for E2, E3, and E4 carriers, respectively—at entry: IHD, 20.0% (n = 14), 21.0% (n = 79), and 21.5% (n = 14); and macroangiopathy, 24.3% (n = 17), 29.3% (n = 110), and 24.6% (n = 16). Apo E polymorphism did not make a significant contribution to multiple logistic regression models designed to identify the factors associated with the occurrence of vascular disease in diabetic patients. CONCLUSION Apo E polymorphism and, notably, the apo E4 allele cannot be universally considered as a particular risk factor for cardiovascular disease in diabetic patients.

The propagation of sound through a one‐dimensional periodic array of water and perspex plates is studied theoretically and experimentally. It is shown that the passbands and stop bands of a scatterer with a finite number of layers correspond to the bands and bandgaps of an infinite ‘‘sonic bandgap crystal.’’ The transmission coefficient of various finite structures is computed and measured as a function of frequency. The analogy with the electronic bandstructure of crystals, and the photonic bandstructure of macroscopic periodic dielectric structures, is found to be a close one. It is shown that the position and width of passbands can easily be engineered. Results are included for a finite ‘‘crystal’’ with a vacancy defect, in which a narrow passband appears in each of the stop bands.

Various studies have linked different genetic single nucleotide polymorphisms (SNPs) to different blood lipids (BL), but whether these "connections" were identified using cross-sectional or longitudinal (i.e., changes over time) designs has received little attention. Cross-sectional and longitudinal assessments of BL [total, high-, low-density lipoprotein cholesterol (TC, HDL, LDL), triglycerides (TG)] and non-genetic factors (body mass index, smoking, alcohol intake) were measured for 2,002 Geneva, Switzerland, adults during 1999-2008 (two measurements, median 6 years apart), and 20 SNPs in 13 BL metabolism-related genes. Fixed and mixed effects repeated measures linear regression models, respectively, were employed to identify cross-sectional and longitudinal SNP:BL associations among the 1,516 (76%) study participants who reported not being treated for hypercholesterolemia at either measurement time. One-third more (12 vs. 9) longitudinal than cross-sectional associations were found [Bonferroni-adjusted two-tailed p < 0.00125 (=0.05/2)/20) for each of the four ensembles of 20 SNP:individual BL associations tested under the two study designs]. There was moderate consistency between the cross-sectional and longitudinal findings, with eight SNP:BL associations consistently identified across both study designs: [APOE.2 and APOE.4 (rs7412 and rs429358)]:TC; HL/LIPC (rs2070895):HDL; [APOB (rs1367117), APOE.2 and APOE.4 (rs7412 and rs429358)]:LDL; [APOA5 (rs2072560) and APOC III (rs5128)]:TG. The results suggest that cross-sectional studies, which include most genome-wide association studies (GWAS), can assess the large majority of SNP:BL associations. In the present analysis, which was much less powered than a GWAS, the cross-sectional study was around 2/3 (67%) as efficient as the longitudinal study.

*Division of Angiology and Hemostasis, Division of Endocrinology, Diabetology and Nutrition, Department of Medical Specialties, GenevaUniversity Hospitals and Faculty of Medicine, Geneva, Switzerland; Hematology Laboratory, §Division of Cardiology, Be´ziers Hospital, Be´ziers;–Hematology Laboratory, Montpellier University Hospital, Montpellier, France; **Division of Laboratory Medicine, Department of Genetics andLaboratory Medicine, Geneva University Hospitals, Geneva; and Division of General Internal Medicine, Geneva University Hospital and Facultyof Medicine, Geneva, SwitzerlandTo cite this article: Fontana P, James R, Barazer I, Berdague´ P, Schved JF, Rebsamen M, Vuilleumier N, Reng JL. Relationship betweenparaoxonase-1 activity, its Q192R genetic variant and clopidogrel responsiveness in the ADRIE study. J Thromb Haemost 2011; 9: 1664–6.

Background Chlamydia trachomatis was previously shown to express a lipoprotein, the macrophage infectivity potentiator (Mip), exposed at the bacterial surface, and able to stimulate human primary monocytes/macrophages through Toll Like Receptor (TLR)2/TLR1/TLR6, and CD14. In PMA-differentiated THP-1 cells the proinflammatory activity of Mip was significantly higher in the absence than in the presence of serum. The present study aims to investigate the ability of different serum factors to attenuate Mip proinflammatory activity in PMA-differentiated THP-1 cells and in primary human differentiated macrophages. The study was also extend to another lipoprotein, the Borrelia burgdorferi outer surface protein (Osp)A. The proinflammatory activity was studied through Tumor Necrosis Factor alpha (TNF-α) and Interleukin (IL)-8 release. Finally, TLR1/2 human embryonic kidney-293 (HEK-293) transfected cells were used to test the ability of the serum factors to inhibit Mip and OspA proinflammatory activity. Results In the absence of any serum and in the presence of 10% delipidated FBS, production of Mip-induced TNF-α and IL-8 in PMA-differentiated THP-1 cells were similar whereas they were significantly decreased in the presence of 10% FBS suggesting an inhibiting role of lipids present in FBS. In the presence of 10% human serum, the concentrations of TNF-α and IL-8 were 2 to 5 times lower than in the presence of 10% FBS suggesting the presence of more potent inhibitor(s) in human serum than in FBS. Similar results were obtained in primary human differentiated macrophages. Different lipid components of human serum were then tested (total lipoproteins, HDL, LDL, VLDL, triglyceride emulsion, apolipoprotein (apo)A-I, B, E2, and E3). The most efficient inhibitors were LDL, VLDL, and apoB that reduced the mean concentration of TNF-α release in Mip-induced macrophages to 24, 20, and 2%, respectively (p < 0.0001). These lipid components were also able to prevent TLR1/2 induced activation by Mip, in HEK-293 transfected cells. Similar results were obtained with OspA. Conclusions These results demonstrated the ability of serum lipids to attenuate proinflammatory activity of bacterial lipoproteins and suggested that serum lipoproteins interact with acyl chains of the lipid part of bacterial lipoproteins to render it biologically inactive.

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.

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.

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.

AIMS: High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. METHODS AND RESULTS: Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. CONCLUSION: In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situations.

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

HDL-cholesterol has a prognostic value for cardiovascular disease at least equivalent to that of LDL-cholesterol. Low levels of HDL-cholesterol constitute the principal dyslipidaemia in the general population, and this will be exacerbated with the predicted explosion in obesity and type 2 diabetes. The latter suggests that low HDL-cholesterol will become of even greater clinical concern. Raising HDL-cholesterol is not as yet considered a primary target for reducing risk of cardiovascular disease, but does constitute an aggravating factor in determining the treatment strategy. Serum HDL-cholesterol levels are variably responsive to lifestyle changes. Pharmacological therapy, which can lead to substantial increases in HDL, may be considered in high risk patients.

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

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.

The increasing longevity of the population, one of the most important issues throughout the planet, is a very complex phenomenon (trait), likely resulting from a variety of environmental determinants interacting with and modulated by genetic mechanisms, mostly devoted to maintenance and repair. In fact, the genes involved in longevity impact upon basic processes such as inflammation, glucose and energy utilization, and oxidative stress. Based on the free radical theory of aging, in the past few years we have focused our attention on an enzyme that protects lipids from peroxidative damage-paraoxonase 1 (PON1). PON1 has been widely investigated, especially for its involvement in atherosclerosis and age-related diseases. In this review, we summarize data on the role played by PON1 on aging and its possible involvement in human longevity, focusing on the relationship between genetic polymorphisms and enzyme activity and its capability to counteract oxidative stress.

ApoA-1 (apolipoprotein A-1) is the main component of HDL (high-density lipoprotein) and stabilizes PON-1 (paraoxonase-1), which prevents lipid peroxidation and oxLDL (oxidized low-density lipoprotein) formation. Autoantibodies against apoA-1 [anti-(apoA-1) IgG] have been found in antiphospholipid syndrome and systemic lupus erythematosous, two diseases with an increased risk of thrombotic events, as well as in ACS (acute coronary syndrome). OxLDL levels are also elevated in these diseases. Whether anti-(apoA-1) IgGs exist in other prothrombotic conditions, such as APE (acute pulmonary embolism) and stroke, has not been studied and their potential association with oxLDL and PON-1 activity is not known. In the present study, we determined prospectively the prevalence of anti-(apoA-1) IgG in patients with ACS (n=127), APE (n=58) and stroke (n=34), and, when present, we tested their association with oxLDL levels. The prevalance of anti-(apoA-1) IgG was 11% in the ACS group, 2% in the control group and 0% in the APE and stroke groups. The ACS group had significantly higher median anti-(apoA-1) IgG titres than the other groups of patients. Patients with ACS positive for anti-(apoA-1) IgG had significantly higher median oxLDL values than those who tested negative (226.5 compared with 47.7 units/l; P

OBJECTIVE To investigate whether genetic polymorphism of paraoxonase (PON1192), an enzyme which protects low density lipoprotein from oxidation, is related to the prevalence of arterial hypertension. METHODS Two groups of carefully selected subjects of both sexes were enrolled and compared. The first group comprised 219 healthy controls (mean age 46.5 +/- 14.7 years) whereas the second comprised 119 hypertensive patients (mean age 47.9 +/- 10.5 years) with untreated essential arterial hypertension. Anthropometric and biochemical parameters were within the normal range in both groups. The PON1192 polymorphism was determined by a polymerase chain reaction-restriction fragment length polymorphism approach. RESULTS In hypertensive patients, a significant increase of the frequency of PON1192RR genotype with respect to healthy controls (14.3 versus 5.0%, P = 0.003) was found. Logistic regression analyses also showed that the PON1192RR genotype was independently associated with a four-fold increase in susceptibility to arterial hypertension (odds ratio = 4.31; 95% confidence interval = 1.63-11.43, P = 0.003). CONCLUSIONS The finding that PON1192RR genotype is associated with a higher prevalence of arterial hypertension may contribute to improving the stratification of cardiovascular risk within a population aged 30-60 years. Determination of the PON1192 polymorphism may help to identify those individuals who are prone to developing cardiovascular diseases at an early stage, suggesting the need for close monitoring of cardiovascular risk factors before the onset of cardiovascular disease.

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382–2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.

Objective: To calibrate Reflotron-measured capillary total cholesterol (cTC) relative to a laboratory-measured venous total cholesterol (vTC) criterion standard for monitoring and screening hypercholesterolemia. Study Design and Setting: Quasi-simultaneous assessment in 1999–2002 of Reflotron cTC and laboratory vTC in a random sample of 4,269 adult residents of Geneva, Switzerland (calibration development subsample n 3,067; validation subsample n 1,172), by means of bias, precision, correlation, sensitivity, and false positive percentage (calculated as 100 specificity) analyses of Reflotron cTC vs. laboratory vTC measures for predicting hypercholesterolemia. Results: Total bias was small (0.26 mmol/L), but there was substantial negative drift in Reflotron cTC (annual biases 0.08, 0.17, 0.27, and 0.60 mmol/L in 1999–2002). Overall, 57% of Reflotron cTC measurements for 894 hypercholesterolemic patients underestimated laboratory vTC (2%, 57%, 71%, and 98% in 1999–2002). Prior to calibration, sensitivity was 73% for the development and 35% for the validation subsample, with false positive at 4% (development) and 0.1% (validation). After calibration, sensitivity was 78% for the development and 37% for the validation subsample, with false positive at 5% (development) and 0.2% (validation). Using 95% upper prediction limits (UPL) for individual vTC values increased sensitivity to 99% and 83% and false positive percentage to 30% and 7% for the development and validation subsamples, respectively. Conclusion: Crude results of Reflotron-measured cTC have poor sensitivity. Instead, 95% UPL can be used for monitoring and screening. Simply adding 0.8 mmol/L to a patient’s observed Reflotron cTC value provides a very good approximation to the 95%

Background and Aim: It has been shown in vitro that the HDL-bound enzyme paraoxonase-1 (PON1) protects LDL against oxidation, and PON1 and PON1 gene polymorphisms may affect the oxidation of HDL particles. The aim of this study was to investigate the associations between in vitro HDL oxidation parameters, endogenous PON1 and PON1 genotypes in families affected by asymptomatic FCHL. Methods and Results: Serum arylesterase (ARE) and PON1 activities, PON1 mass, PON1 genotypes and the kinetics of CuSO4-induced HDL oxidation in vitro were measured in 150 members of FCHL families free of clinical CAD. At univariate analysis, log PON1/apoA-I and the PON1 mass/apoA-I ratio significantly correlated with lag time, maximum diene formation and the propagation rate. The oxidation parameters also correlated with PON1 genotypes. Multivariate analysis showed that the associations between PON1 mass/unit apoA-I and the oxidation parameters were independent of the other variables. The lag time of HDL oxidation was also associated with the PON1 genotype 192QR. Conclusions: Endogenous PON1 may have protective effects on the different stages of HDL oxidation in the members of families affected by FCHL. This protective effect is independent of other biochemical factors, but may be influenced by the PON1 gene polymorphism. The endogenous PON1 content of HDL seems to be an important determinant of the anti-atherogenicity of this lipoprotein.

Aims To analyse the association of smoking with paraoxonase (PON1) in Type 2 diabetic patients. Methods Type 2 diabetic patients were recruited independently in two centres (Ancona, Italy and Geneva, Switzerland) and serum PON1 mass and activities were assayed. Current smoking status was established and its association with serum PON1 analysed. Results Type 2 diabetic patients who smoked had significantly lower serum PON1 mass and activity. This was evident in both groups of patients, even though Swiss patients were composed of coronary patients. Multivariate analyses established that smoking was an independent determinant of serum PON1 status. Conclusions Smoking is associated with reduced serum levels of the antioxidant enzyme, PON1, even against an already unfavourable background of diabetes and coronary disease. It suggests that a combination of smoking and diabetes may be particularly deleterious for PON1 and consequently for the anti-oxidant capacity of high-density lipoproteins.

Apolipoprotein E (apo E) is a normal component of several classes of plasma lipoproteins. Apo E phenotypes are closely related to total cholesterol, low density lipoprotein (LDL)-cholesterol and apo B concentration. The apo E 2/2 phenotype is related to the type III hyperlipoproteinemia due to the defective binding of apo E-2 to the hepatic receptors. The apo E 4/4 phenotype has been reported to be present in most elderly people suffering from the Alzheimer disease, and is associated with increased risk of coronary heart disease and Creutzfeld-Jakob disease. Therefore, apo E phenotyping is essential. The method described here uses a precast immobilized pH gradient, avoids time-consuming separation of lipoproteins from plasma, needs no pretreatment with neuraminidase and involves highly sensitive enhanced chemiluminescence for visualization. Therefore it has many advantages over previously published methods.

Increasing evidence supports the involvement of inflammation in the early phases of atherogenesis. Recruitment of leukocytes within the vascular wall, controlled by chemokines, is an essential process in the development of this common disease. In this study, we report that blocking a chemokine pathway in vivo with the CC chemokine antagonist Met-RANTES reduces the progression of atherosclerosis in a hypercholesterolemic mouse model. The reduction of lesions was correlated with a diminution of expression of several major chemokines and chemokine receptors, a decrease in leukocyte infiltration, and an increase of collagen-rich atheroma, features associated with stable atheroma. Treatment was well tolerated and serum lipid profiles were not affected. Whereas genetically engineered mice with deletion of either a CC chemokine or its receptor have demonstrated resistance to disease, to our knowledge, this is the first demonstration that treatment with a chemokine receptor antagonist limits the progression of atherosclerosis in vivo. Thus, our findings indicate that blockade of chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce the evolution of this common disease.

Background and objectives Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work demonstrated that the systemic administration of IL-33 reduces the development of atherosclerosis in the apolipoprotein E-deficient (ApoE -/- ) mouse model of the disease by induction of a Th1-to-Th2 shift. However, the role of endogenous IL-33 in the atherogenesis remains elusive. Materials and methods Atherosclerosis was induced in 10 week-old ApoE -/- , IL-33 -/- ApoE -/- and ST2 -/- ApoE -/- mice by feeding a high-cholesterol diet (1.25%, no cholate) for 10 weeks. Additionally, a group of ApoE -/- mice were injected with a neutralising anti-ST2 antibody or an isotype control during the period of the diet. The atherosclerotic lesion development was measured with Oil Red O in the thoracic-abdominal aorta and in the aortic sinus. The mRNA levels of several cytokines, including IL-6, IFNγ, IL-17, IL-5 and IL-10 were assessed in the aorta and in in vitro -stimulated lymph node cells. Results We observed no differences in lipid-staining area in the aortas of IL-33 -/- ApoE -/- mice (8.84 ± 0.97; mean ± SEM; n = 9–25), ST2 -/- ApoE -/- mice (6.95 ± 0.78), ApoE -/- mice untreated (7.05 ± 0.78), ApoE -/- mice injected with either the neutralising anti-ST2 antibody (6.08 ± 0.79) or the isotype control (6.16 ± 0.86) after high-cholesterol diet feeding. Similar results were obtained in the aortic sinus compared to ApoE -/- controls. Total serum cholesterol and triglyceride levels were not different compared to ApoE -/- controls. IL-33 expression in aortic tissue was comparable in ApoE -/- and ST2 -/- ApoE -/- mice and absent in IL-33 -/- ApoE -/- mice. There was no difference in the transcript levels of inflammatory cytokines in the aorta and in in vitro -stimulated lymph node cells. Conclusions These data indicate that in contrast to the anti-atherosclerotic effect of systemically administered recombinant IL-33, the endogenously produced cytokine and its receptor do not significantly influence the severity of atherosclerosis in ApoE-deficient mice fed with a high-cholesterol diet.

The inverse association between moderate drinking and coronary heart disease mortality is well established. This study was performed to investigate the kinetics of the alcohol-induced increases in apo A-1, HDL cholesterol, and paraoxonase (PON) activity, as well as to study whether the alcohol-induced increases in PON activity differ within different PON polymorphisms, and to investigate whether moderate alcohol consumption has similar effects on the outcome measures in postmenopausal women as in middle-aged men.In a randomized, diet-controlled, crossover study, 10 middle-aged men and 9 postmenopausal women, all apparently healthy, nonsmoking, and moderate alcohol drinkers, consumed beer or no-alcohol beer (control) with evening dinner during two successive periods of 3 weeks. During the beer period, alcohol intake equaled 40 and 30 g/day for men and women, respectively. The total diet was supplied to the subjects and had essentially the same composition during these 6 weeks. Before each treatment was a 1-week washout period, in which the subjects were not allowed to drink alcoholic beverages.Moderate alcohol consumption significantly increased serum apo A-I level after 5 days (3.7%, p0.05); after 10 days, serum HDL cholesterol level was increased (6.8%, p0.001), and after 15 days serum PON activity was increased (3.7%, p0.05), all compared with no alcohol consumption. Gene polymorphisms did not modulate the alcohol effect on PON.Serum apo A-I, HDL cholesterol, and PON activity were significantly increased during 3 weeks of moderate alcohol consumption as compared with no alcohol consumption. Moreover, the results suggest that there is a sequence in induction of these parameters. After an increase in apo A-I, HDL cholesterol is increased followed by an increase in PON activity. Increased serum HDL cholesterol level and PON activity may be a mechanism of action not only in healthy middle-aged men but also in postmenopausal women, underlying the reduced coronary heart disease risk in moderate drinkers.

Increased levels of physical activity may improve the lipid profile, but is this effect identical across apolipoprotein E ( apoE ) genotypes? A population-based cross-sectional survey conducted from 1999 to 2000 included 1708 randomly selected men and women aged 35 to 74 years. A validated physical activity questionnaire measured, for each participant, the total energy expenditure and its percentage used in high-intensity activities (%high-intensity activity), eg, brisk walking and sports. The effects of the apoE× %high-intensity activity interaction on the lipid profile were investigated by using multiple linear regression models. Among men, increased %high-intensity activity had greater protective effects in the apoE4 group regarding (1) high density lipoprotein (HDL) cholesterol ( P apoE2 (interaction P =0.05) or apoE3 (interaction P P apoE3 group (interaction P =0.07). A 10% increase of %high-intensity activity by an apoE4 man would correspond with a 0.07-mmol/L increase in HDL cholesterol and a −0.15-mmol/L decrease in triglycerides. Among women, only the protective effects of physical activity on HDL cholesterol in the apoE4 group versus the apoE2 group was statistically significant. Spending a larger fraction of the total energy expenditure in high-intensity activities may counteract the atherogenic effects of the ε 4 allele on the lipid profile.

Background: The inverse association between moderate drinking and coronary heart disease mortality is well established. This study was performed to investigate the kinetics of the alcohol-induced increases in apo A-1, HDL cholesterol, and paraoxonase (PON) activity, as well as to study whether the alcohol-induced increases in PON activity differ within different PON polymorphisms, and to investigate whether moderate alcohol consumption has similar effects on the outcome measures in postmenopausal women as in middle-aged men. Methods: In a randomized, diet-controlled, crossover study, 10 middle-aged men and 9 postmenopausal women, all apparently healthy, nonsmoking, and moderate alcohol drinkers, consumed beer or no-alcohol beer (control) with evening dinner during two successive periods of 3 weeks. During the beer period, alcohol intake equaled 40 and 30 g/day for men and women, respectively. The total diet was supplied to the subjects and had essentially the same composition during these 6 weeks. Before each treatment was a 1-week washout period, in which the subjects were not allowed to drink alcoholic beverages. Results: Moderate alcohol consumption significantly increased serum apo A-I level after 5 days (3.7%, p < 0.05); after 10 days, serum HDL cholesterol level was increased (6.8%, p < 0.001), and after 15 days serum PON activity was increased (3.7%, p < 0.05), all compared with no alcohol consumption. Gene polymorphisms did not modulate the alcohol effect on PON. Conclusions: Serum apo A-I, HDL cholesterol, and PON activity were significantly increased during 3 weeks of moderate alcohol consumption as compared with no alcohol consumption. Moreover, the results suggest that there is a sequence in induction of these parameters. After an increase in apo A-I, HDL cholesterol is increased followed by an increase in PON activity. Increased serum HDL cholesterol level and PON activity may be a mechanism of action not only in healthy middle-aged men but also in post-menopausal women, underlying the reduced coronary heart disease risk in moderate drinkers. Chemicals/CAS: Apolipoprotein A-I; Aryldialkylphosphatase, EC 3.1.8.1; Cholesterol, HDL; Esterases, EC 3.1.

Purpose: Apolipoprotein M (apoM) is a plasma lipoprotein that mainly associates with high-density lipoproteins (HDL) and that serves as a carrier of the bioactive lipid Sphingosine-1-Phosphate (S1P). Recent studies indicate that S1P binding to G-protein-coupled receptors, known as S1P-receptors, in the heart activates signalling pathways promoting cardiomyocyte survival, but downstream targets are largely unknown. Here, we investigate the putative role of the apoM-S1P axis in relation to cardioprotection against ischemia/reperfusion (IR) injury. Methods and Results: ApoM transgenic (Apom-Tg) mice, in which plasma S1P is increased by >250%, and wild-type (WT) mice were subjected to 30 min of left coronary artery ligation and 24 hrs reperfusion in vivo. We found a reduction of infarct size in Apom-Tg mice (15±1%) in comparison with WT mice (29±4%, N=8-9, p

Purpose: Exogenous administration of sphingosine-1-phosphate (S1P) alone, or as part of high density lipoprotein, protects against myocardial infarction. S1P-induced cardioprotection targets the inhibition of the mitochondrial permeability transition pore via mechanisms that remain unclear. In the cell, the endogenous production of S1P from sphingosine is dependent on the activation of sphingosine kinases (SphK) 1 and 2. These two kinases play a role in cardioprotection against ischemia-reperfusion (IR) injury. Therefore, we hypothesised that the cardioprotective effect of exogenous S1P requires the activation of endogenous S1P via SphK. Methods: Isolated cardiomyocytes from adult wildtype mice were exposed to 2 hours of simulated ischemia (SI) in the presence of S1P (10nM) with/without N,N-dimethylsphingosine (DMS, a SphK1 and 2 inhibitor, 10μM) or SKI (a specific SphK1 inhibitor, 15μM). Cell viability was assessed using trypan blue staining and normalised to the normoxic control. Isolated perfused hearts from adult wildtype mice were exposed to 35 minutes of global ischemia followed by 45 minutes of reperfusion (IR) in the presence of S1P (10nM) with/without SKI (10μM). Infarct size (IS) was assessed using tripheyltetrazolium chloride staining and SphK1 activity using a specific biochemical fluorescence based assay kit. Both parameters were normalised to the IR control. Results: In isolated cardiomyocytes, viability under normoxic conditions was 76±1%. SI reduced viability to 52±1% (p< 0.001 vs. normoxia). Pre-treatment with S1P restored the viability to 75±1% (p

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.

International audience; Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.

The close association between diabetes and cardiovascular disease suggests that current predictions of a massive increase in the prevalence of type 2 diabetes foreshadow an equally daunting rise in the incidence of vascular disease. The limited cardiovascular benefits obtained by glucose-lowering treatments, although perhaps not surprising, indicate that other cardiovascular risk factors must be given serious consideration as therapeutic targets. The impressive reductions in the number of vascular events observed in diabetic patients, albeit in small patient populations, participating in various drug trials amply justify such an approach. A necessary prerequisite, however, is a clear understanding of the clinical importance of individual risk factors to the occurrence of vascular disease in type 2 diabetic patients. This would appear essential for defining treatment strategies in the face of a bewildering array of potential therapeutic targets. The present review considers recent studies that have assessed the predictive value of risk factors against a diabetic background.

Apolipoprotein A-1 (Apo A-1) is the major protein fraction of high-density lipoproteins whose protective role in the cardiovascular system has been established. Anti-Apo A-1 IgG autoantibodies were described in autoimmune disorders such as systemic lupus erythematosus and antiphospholipid syndrome and might be involved in the genesis of arterial and venous thrombotic events. Anti-Apo A-1 IgG have been reported in myocardial infarction (MI) …