Sphingosine-1-phosphate as a key player of insulin secretion induced by high-density lipoprotein treatment.

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High-density lipoprotein (HDL) has been proposed to improve ß-cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine-1-phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic ß-cells and to determine its mechanisms. Primary cultures of ß-cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL-S1P) isolated from T2DM patients was analyzed and correlated to the HDL-induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose-stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL-S1P was strongly correlated to its pro-secretory capacity (r = 0.633, p = 0.005), HDL-cholesterol and apolipoprotein AI levels were not. HDL-induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in ß-cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL-S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra- and extra-cellular sources of S1P independently of an effect on insulin biosynthesis genes.

Low Serum Paraoxonase-1 Activity Associates with Incident Cardiovascular Disease Risk in Subjects with Concurrently High Levels of High-Density Lipoprotein Cholesterol and C-Reactive Protein.

Paroxonase-1 (PON1) is a key enzyme that inhibits low-density lipoprotein oxidation and consequently atherogenesis. Here, we assessed whether low serum PON1 activity associates with incident cardiovascular disease (CVD) in subjects with high levels of high-density cholesterol (HDL-C) and C-reactive protein (CRP), a marker of low-grade systemic inflammation. Cox proportional-hazards modeling of incident CVD risk (11 years mean follow-up) adjusted for relevant clinical and biomarker covariates was performed on a population-based study (N = 7766) stratified into three groups: low CRP-(LR; event rate 4.9%); low HDL-C/high CRP-(HR1; event rate 14.4%); and high HDL-C/high CRP-(HR2; event rate 7.6%). Modeling results for PON1 activity in HR2 were significant and robust (hazard ratio/SD unit-0.68, 95% CI 0.55-0.83, p = 0.0003), but not so for LR and HR1. Analyses in HR2 of the interaction of PON1 with HDL-C, apoA-I, apoA-II, and apoE levels were significant only for PON1 with apoE (hazard ratio-1.77, 95% CI 1.29-2.41, p = 0.0003). Subsequent subgroup analysis revealed inverse risk dependence for apoE at low PON1 levels. In conclusion, in a population-based study of subjects with concurrently high HDL-C and CRP levels, low serum PON1 activity associates with incident CVD risk with risk accentuated at low apoE levels.

Circulating Lactonase Activity but Not Protein Level of PON-1 Predicts Adverse Outcomes in Subjects with Chronic Kidney Disease.

The burden of cardiovascular disease and death in chronic kidney disease (CKD) outpaces that of the other diseases and is not adequately described by traditional risk factors alone. Diminished activity of paraoxonase (PON)-1 is associated with increased oxidant stress, a common feature underlying the pathogenesis of CKD. We aimed to assess the prognostic value of circulating PON-1 protein and PON lactonase activity on adverse clinical outcomes across various stages and etiologies of CKD. Circulating PON-1 protein levels and PON lactonase activity were measured simultaneously in patients with CKD as well as a cohort of apparently healthy non-CKD subjects. Both circulating PON-1 protein levels and PON lactonase activity were significantly lower in CKD patients compared to the non-CKD subjects. Similarly, across all stages of CKD, circulating PON-1 protein and PON lactonase activity were significantly lower in patients with CKD compared to the non-CKD controls. Circulating PON lactonase activity, but not protein levels, predicted future adverse clinical outcomes, even after adjustment for traditional risk factors. The combination of lower circulating protein levels and higher activity within the CKD subjects were associated with the best survival outcomes. These findings demonstrate that diminished circulating PON lactonase activity, but not protein levels, predicts higher risk of future adverse clinical outcomes in patients with CKD.

HDL protects against myocardial ischemia reperfusion injury via miR-34b and miR-337 expression which requires STAT3.

PURPOSE: High density lipoprotein (HDL) protects against myocardial infarction via mechanisms that remain unclear. STAT3 (signal transducer and activator of transcription 3) plays a key role in HDL-induced cardioprotection. In the heart, microRNAs (miRNAs) are involved in ischemia reperfusion injury. We therefore investigated whether the cardioprotective effect of HDL modulates miRNAs as a downstream target of STAT3 activation. METHODS: STAT3 cardiomyocyte deficient mice (STAT3-KO) and wildtype littermates (STAT3-WT) were submitted to left coronary ligature and reperfused (IR) with or without injection of HDL. Infarct size (IS) was determined and cardiac miRNA expression was evaluated after reperfusion in sham, IR and IR+HDL hearts by microarray analysis. In vitro, neonatal rat ventricular cardiomyocytes were submitted to hypoxia with or without HDL incubation. Cell viability and miRNA expression were analysed. RESULTS: In vivo, HDL reduced IS from 40.5±4.3% to 24.4±2.1% (p<0.05) in STAT3-WT mice. HDL failed to protect in STAT3-KO mice. In STAT3-WT mice, both miR-34b and miR-337 were increased in IR compared to sham and IR+HDL groups (p<0.05). These miRNAs were not modulated in STAT3-KO mice. In vitro, incubation with HDL improved cell viability against hypoxia (p<0.05). The expression of miR-34b and miR-337 was increased by hypoxia and reduced by HDL treatment (p<0.05). In cardiomyocytes transfected with miRNA mimics, HDL failed to improve cell viability against hypoxia. CONCLUSIONS: Our study, performed both in vivo and in vitro, delineates a novel cardioprotective signalling pathway activated by HDL, involving STAT3-mediated decrease of miR-34b and miR-337 expression.

Serum paraoxonase 1 activity is paradoxically maintained in nonalcoholic fatty liver disease despite low HDL cholesterol.

Nonalcoholic fatty liver disease (NAFLD) is characterized by low HDL cholesterol, but the activity of the HDL-associated antioxidative enzyme paraoxonase-1 (PON-1) remains unclear. To determine the association of PON-1 with suspected NAFLD, we measured serum enzyme activity in 7,622 participants of the Prevention of Renal and Vascular End-Stage Disease cohort. A fatty liver index (FLI) ≥60, a proxy of NAFLD, was present in 2,083 participants (27.3%) and coincided with increased prevalence of T2D, metabolic syndrome (MetS), (central) obesity, elevated triglycerides, and low HDL cholesterol (all P < 0.001). In men and women combined, serum PON-1 activity did not vary according to elevated FLI (P = 0.98), whereas in men with elevated FLI PON-1 activity was increased (P = 0.016). In multivariable linear regression analyses (adjusted for age, sex, T2D, MetS, alcohol use, and smoking), PON-1 activity was unexpectedly associated with elevated FLI (ß = 0.083; P < 0.001). In a sensitivity analysis (n = 5,126) that excluded subjects with positive cardiovascular history, impaired estimated glomerular filtration rate, elevated urinary albumin excretion, and drug use, PON-1 activity was also independently associated with elevated FLI (ß = 0.045; P = 0.017). These results indicate that PON-1 is paradoxically maintained and may even be increased in NAFLD despite inverse associations with metabolic disorders and low HDL cholesterol.

Serum paraoxonase-1 activity is associated with light to moderate alcohol consumption: the PREVEND cohort study.

Background: Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL)-associated enzyme with antioxidative properties, which may protect against the development of cardiovascular disease. Alcohol consumption increases HDL cholesterol, but the extent to which alcohol consumption gives rise to higher serum PON-1 activity is uncertain. Objective: In a population-based study, we determined the relation of serum PON-1 activity with alcohol consumption when taking account of HDL cholesterol and apolipoprotein A-I (apoA-I), its major apolipoprotein. Design: A cross-sectional study was performed in 8224 participants of the Prevention of Renal and Vascular End-Stage Disease (PREVEND) cohort. Alcohol consumption was categorized as 1) no/rarely (25.3%); 2) 0.1-10 g/d (49.3%); 3) 10-30 g/d (20.1%); and 4) >30 g/d (5.2%) with 1 drink equivalent to 10 g alcohol. Serum PON-1 activity was measured as its arylesterase activity (phenyl acetate as substrate). Results: Median serum PON-1 activity was 50.8, 53.1, 54.4, and 55.7 U/L in the 4 categories of alcohol consumption, respectively (P < 0.001). Its increase paralleled the increments in HDL cholesterol and apoA-I. Notably, there was no further increase in PON-1 activity, HDL cholesterol, and apoA-I when alcohol consumption was increased from 10-30 g/d to >30 g/d. Multivariable linear regression analysis demonstrated that PON-1 activity was related to alcohol consumption independently from clinical covariates, high sensitivity C-reactive protein, and lipid concentrations, including HDL cholesterol (P < 0.001 for each category of alcohol consumption with no alcohol consumption as the reference category). Notably, as inferred from standardized ß-coefficients, there was no difference in PON-1 activity between 10-30 g alcohol/d and >30 g alcohol/d. Conclusions: Alcohol consumption is associated with an increase in serum PON-1 activity, but its effect seems to reach a plateau with alcohol consumption of 10-30 g/d.

Differential Association of Cx37 and Cx40 Genetic Variants in Atrial Fibrillation with and without Underlying Structural Heart Disease.

Atrial fibrillation (AF) appears in the presence or absence of structural heart disease. The majority of foci causing AF are located near the ostia of pulmonary veins (PVs), where cardiomyocytes and vascular smooth muscle cells interdigitate. Connexins (Cx) form gap junction channels and participate in action potential propagation. Genetic variants in genes encoding Cx40 and Cx37 affect their expression or function and may contribute to PV arrhythmogenicity. DNA was obtained from 196 patients with drug-resistant, symptomatic AF with and without structural heart disease, who were referred for percutaneous catheter ablation. Eighty-nine controls were matched for age, gender, hypertension, and BMI. Genotyping of the Cx40 -44G > A, Cx40 +71A > G, Cx40 -26A > G, and Cx37 1019C > T polymorphisms was performed. The promoter A Cx40 polymorphisms (-44G > A and +71A > G) showed no association with non-structural or structural AF. Distribution of the Cx40 promoter B polymorphism (-26A > G) was different in structural AF when compared to controls (p = 0.03). There was no significant difference with non-structural AF (p = 0.50). The distribution of the Cx37 1019C > T polymorphism was different in non-structural AF (p = 0.03) but not in structural AF (p = 0.08) when compared to controls. Our study describes for the first time an association of drug-resistant non-structural heart disease AF with the Cx37 1019C > T gene polymorphism. We also confirmed the association of the Cx40 - 26G > A polymorphism in patients with AF and structural disease.

High-density lipoprotein from end-stage renal disease patients exhibits superior cardioprotection and increase in sphingosine-1-phosphate.

BACKGROUND: Chronic kidney disease (CKD) exacerbates the risk of death due to cardiovascular disease (CVD). Modifications to blood lipid metabolism which manifest as increases in circulating triglycerides and reductions in high-density lipoprotein (HDL) cholesterol are thought to contribute to increased risk. In CKD patients, higher HDL cholesterol levels were not associated with reduced mortality risk. Recent research has revealed numerous mechanisms by which HDL could favourably influence CVD risk. In this study, we compared plasma levels of sphingosine-1-phosphate (S1P), HDL-associated S1P (HDL-S1P) and HDL-mediated protection against oxidative stress between CKD and control patients. METHODS: High-density lipoprotein was individually isolated from 20 CKD patients and 20 controls. Plasma S1P, apolipoprotein M (apoM) concentrations, HDL-S1P content and the capacity of HDL to protect cardiomyocytes against doxorubicin-induced oxidative stress in vitro were measured. RESULTS: Chronic kidney disease patients showed a typical profile with significant reductions in plasma HDL cholesterol and albumin and an increase in triglycerides and pro-inflammatory cytokines (TNF-alpha and IL-6). Unexpectedly, HDL-S1P content (P = .001) and HDL cardioprotective capacity (P = .034) were increased significantly in CKD patients. Linear regression analysis of which factors could influence HDL-S1P content showed an independent, negative and positive association with plasma albumin and apoM levels, respectively. DISCUSSION: The novel and unexpected observation in this study is that uremic HDL is more effective than control HDL for protecting cardiomyocytes against oxidative stress. It is explained by its higher S1P content which we previously demonstrated to be the determinant of HDL-mediated cardioprotective capacity. Interestingly, lower concentrations of albumin in CKD are associated with higher HDL-S1P.

Serum paraoxonase-1 activity is inversely related to free thyroxine in euthyroid subjects: The PREVEND Cohort Study.

BACKGROUND: Low-normal thyroid function within the euthyroid range has been suggested to enhance atherosclerosis susceptibility. Paraoxonase-1 (PON-1) may protect against atherosclerotic cardiovascular disease development by attenuating oxidative stress. We evaluated relationships of PON-1 with thyroid stimulating hormone (TSH), free T4 , free T3 , lipids and apolipoprotein (apo)A-I in euthyroid subjects, and assessed whether such relationships are modified in the context of the metabolic syndrome (MetS). MATERIALS AND METHODS: Serum PON-1 activity (arylesterase activity), TSH, free T4 , free T3 , lipids and apoA-I was measured in 2206 euthyroid subjects (aged 28-75 years; 1138 men (age 49 ± 13 years) and 1068 women (age 46 ± 12 years), recruited from the general population (PREVEND cohort). RESULTS: In age- and sex-adjusted analysis, PON-1 activity (divided into tertiles) was positively related to TSH (ß = -0.045, P = .036) and inversely to free T4 (ß = -0.042, P = .050) but not to free T3 (ß = -0.027, P = .20). PON-1 activity was positively related to total cholesterol, non-HDL cholesterol and triglycerides, as well as to HDL cholesterol and apoA-I (P < .01 to <.001). The inverse relationship of PON-1 activity with free T4 remained present after adjustment for lipids and other potential confounders (ß = -0.066, P = .002), but the positive relationship with TSH lost significance (ß = 0.034, P = .11). The inverse relationship of PON-1 activity with free T4 was not different in subjects with vs without MetS (P = .94), nor modified by the presence of its individual components (P ≥ .22 for each). CONCLUSIONS: Serum PON-1 activity is inversely associated with free T4 in euthyroid subjects, suggesting that low-normal thyroid function may affect PON-1 regulation.

The inverse association of HDL-cholesterol with future risk of hypertension is not modified by its antioxidant constituent, paraoxonase-1: The PREVEND prospective cohort study.

BACKGROUND AND AIMS: High-density lipoprotein cholesterol (HDL-C), an established risk marker for atherosclerotic cardiovascular disease (CVD), has been shown to be inversely and independently associated with incident hypertension. Paraoxonase-1 (PON-1) is an HDL-bound esterase enzyme associated with CVD, but its relationship with incident hypertension has not been previously investigated. We aimed at evaluating the prospective association between PON-1 and hypertension risk. METHODS: PON-1 arylesterase activity was measured in serum at baseline in 3988 participants without pre-existing hypertension in the Prevention of Renal and Vascular End-stage Disease (PREVEND) prospective population-based study. During a median follow-up of 10.7 years, 1206 participants developed hypertension. RESULTS: In age- and sex-adjusted analysis, the hazard ratio (95% CI) for incident hypertension per 1 standard deviation increase in PON-1 was 1.01 (0.96-1.07; p = 0.656), which remained non-significant after adjustment for several established hypertension risk factors and other potential confounders (0.99, 0.93 to 1.05; p = 0.764). The association was also non-existent on further adjustment for HDL-C (1.00 (0.94-1.06; p = 0.936)) and did not importantly vary across several clinical subgroups. In analyses in the same set of participants, HDL-C was continuously inversely and independently associated with hypertension risk; the association persisted after further adjustment for PON-1 activity and was not modified by PON-1 activity. CONCLUSIONS: In this Caucasian cohort of men and women, HDL-C, but not its anti-oxidant constituent - PON-1, is inversely, continuously and independently associated with future risk of hypertension. The association is independent of and not modified by PON-1.

Incident type 2 diabetes is associated with HDL, but not with its anti-oxidant constituent - paraoxonase-1: The prospective cohort PREVEND study.

OBJECTIVE: High-density lipoprotein cholesterol (HDL-C) is an established risk marker for cardiovascular disease and consistently associated with type 2 diabetes risk. Serum paraoxonase-1 (PON-1) - an anti-oxidant constituent of HDL - is inversely associated with cardiovascular disease risk, but its relationship with incident type 2 diabetes is uncertain. We aimed to investigate the prospective association between PON-1 and type 2 diabetes risk. METHODS: PON-1 was measured as its arylesterase activity at baseline in the Prevention of Renal and Vascular End-stage Disease (PREVEND) prospective study of 5947 predominantly Caucasian participants aged 28-75years with no pre-existing diabetes, that recorded 500 type 2 diabetes cases during a median follow-up of 11.2years. RESULTS: Serum PON-1 was positively correlated with HDL-C (r=0.17; P<0.001). In analyses adjusted for conventional diabetes risk factors, the hazard ratio (95% CI) for type 2 diabetes per 1 standard deviation increase in PON-1 was 1.07 (0.98 to 1.18; P=0.13), which remained non-significant (1.02 (0.93 to 1.12) P=0.65) after additional adjustment for potential confounders. The association was unchanged on further adjustment for HDL-C (1.05 (0.96 to 1.15; P=0.29). However, in subsidiary analyses in the same set of participants, serum HDL-C concentration was inversely and independently associated with risk of type 2 diabetes. CONCLUSIONS: Incident type 2 diabetes is associated with HDL cholesterol but not with its anti-oxidant constituent - PON-1 - in a large cohort of apparently healthy men and women. The current data question the importance of PON-1 activity for the development of diabetes.

Self-Observation and Peer Feedback as a Faculty Development Approach for Problem-Based Learning Tutors: A Program Evaluation.

PROBLEM: Good teaching requires spontaneous, immediate, and appropriate action in response to various situations. It is even more crucial in problem-based learning (PBL) tutorials, as the tutors, while directing students toward the identification and attainment of learning objectives, must stimulate them to contribute to the process and provide them with constructive feedback. PBL tutors in medicine lack opportunities to receive feedback from their peers on their teaching strategies. Moreover, as tutorials provide little or no time to stop and think, more could be learned by reflecting on the experience than from the experience itself. We designed and evaluated a faculty development approach to developing PBL tutors that combined self-reflection and peer feedback processes, both powerful techniques for improving performance in education. INTERVENTION: We developed an observation instrument for PBL facilitation to be used both by tutors to self-observe and reflect on own teaching strategies and by peers to observe and provide feedback to tutors. Twenty PBL sessions were video-recorded. Tutors completed the instrument immediately after their PBL session and again while watching their video-recorded session (self-observation). A group of three observers completed the instrument while watching each recorded session and provided feedback to each tutor (peer observation and feedback). We investigated tutors' perceptions of the feasibility and acceptability of the approach and gathered data on its effectiveness in enhancing tutors' facilitation skills. CONTEXT: The preclinical medical curriculum at the University of Geneva is essentially taught by PBL. A new program of faculty development based on self-observation and peer feedback was offered to voluntary tutors and evaluated. OUTCOME: Our results suggest that self-observation and peer feedback, supported by an instrument, can be effective in enhancing tutors' facilitation skills. Reflection on self-observation raised teachers' awareness of the effectiveness of the strategies they used to foster student learning. This motivated a need to change their teaching practice. However, for the changes to become operative, peer feedback was required, providing the cues and strategies needed to improve the facilitation skills. LESSONS LEARNED: Peer coaching was considered feasible and useful to improve tutors' facilitation skills. Evaluating the program made it possible to assess tutors' needs and the reasons underlying their difficulties, and this in turn provided the basis for advanced workshops. Nonetheless, aspects related to logistics and the time constraints of such an individualized approach, as well as the cultural appropriation of peer coaching, might be obstacles that need to be addressed.

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

BACKGROUND: 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. MATERIALS AND METHODS: In a case-control study, HDL was isolated from 12 FH patients with and without statin treatment and from 12 healthy controls. The HDL-S1P content and the capacity of HDL to protect cardiomyocytes against oxidative stress in vitro were measured. RESULTS: HDL-associated S1P was significantly correlated with cell protection, but not with HDL-cholesterol or apolipoprotein AI. The latter did not correlate with HDL-mediated cell protection. Neither the HDL-S1P content nor HDL protective capacity differed between nontreated FH patients and controls. The relative amounts of apolipoprotein AI and apolipoprotein M were similar between controls and FH patients. Statin treatment had no effect on any of these measures. CONCLUSIONS: The FH environment is not detrimental to HDL-S1P content or HDL-S1P-mediated cell protection. Statin treatment does not modulate HDL function in this regard.

Pharmacological Intervention to Modulate HDL: What Do We Target?

The cholesterol concentrations of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) have traditionally served as risk factors for cardiovascular disease. As such, novel therapeutic interventions aiming to raise HDL cholesterol have been tested in the clinical setting. However, most trials led to a significant increase in HDL cholesterol with no improvement in cardiovascular events. The complexity of the HDL particle, which exerts multiple physiological functions and is comprised of a number of subclasses, has raised the question as to whether there should be more focus on HDL subclass and function rather than cholesterol quantity. We review current data regarding HDL subclasses and subclass-specific functionality and highlight how current lipid modifying drugs such as statins, cholesteryl ester transfer protein inhibitors, fibrates and niacin often increase cholesterol concentrations of specific HDL subclasses. In addition this review sets out arguments suggesting that the HDL3 subclass may provide better protective effects than HDL2.

Abca1 deficiency protects the heart against myocardial infarction-induced injury.

BACKGROUND AND AIMS: We explored the role of ATP-binding cassette transporter A1 (Abca1), in post-myocardial infarction (MI) cardiac injury. METHODS: In Abca1(-/-) mice, wild type (WT) mice, and WT mice transplanted with Abca1(-/-) or WT bone marrow, an MI was induced in vivo. Furthermore, an ex vivo MI was induced in isolated Abca1(-/-) and WT hearts. RESULTS: Twenty-four hours and two weeks after in vivo MI induction, MI size was reduced in Abca1(-/-) (-58%, p = 0.007; -59%, p = 0.03) compared to WT. Ex vivo MI induction showed no effect of Abca1(-/-) on infarct size. Interestingly, two weeks after MI, Abca1(-/-) mice showed higher circulating levels of B-cells (+3.0 fold, p = 0.02) and T-cells (+4.2 fold, p = 0.002) compared to WT. Bone marrow-specific Abca1(-/-) tended to reduce infarct size (-43%, p = 0.12), suggesting a detrimental role for hematopoietic Abca1 after MI. CONCLUSIONS: Although Abca1 has a protective role in atherosclerosis, it exerts detrimental effects on cardiac function after MI.

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.

Serum paraoxonase-1 activity and risk of incident cardiovascular disease: The PREVEND study and meta-analysis of prospective population studies.

BACKGROUND: Paraoxonase-1 (PON-1) has been suggested to be associated with cardiovascular disease (CVD) risk, however, aspects of the association, such as shape and independence from conventional risk factors are still uncertain. We aimed to assess the association of PON-1 with CVD risk and determine its potential utility for CVD risk prediction. METHODS: PON-1 was measured as its arylesterase activity at baseline in the PREVEND prospective study of 6902 participants. RESULTS: During a mean follow-up of 9.3 years, 730 CVD events were recorded. Serum PON-1 was weakly correlated with several cardiovascular risk markers including high-density lipoprotein cholesterol (HDL-C) (r = 0.18; P < 0.001) and was approximately log-linearly associated with CVD risk. In analyses adjusted for conventional risk factors, the hazard ratio (95% CI) for CVD per 1 standard deviation (SD) increase in loge PON-1 was 0.92 (0.85-0.99; P = 0.020), which remained persistent after additional adjustment for potential confounders 0.93 (0.86-0.99; P = 0.037). The association was attenuated on further adjustment for HDL-C 0.95 (0.88-1.02; P = 0.153). In a meta-analysis of 6 population-based prospective studies involving 15 064 participants and 2958 incident CVD outcomes, the pooled multivariable adjusted (including HDL-C) relative risk (95% CI) for CVD was 0.95 (0.90-1.02; P = 0.138) per 1 SD increase in PON-1 values. Adding PON-1 to a CVD risk prediction model containing conventional risk factors did not improve the C-index or net reclassification. CONCLUSIONS: There is an approximately log-linear inverse association between PON-1 activity and CVD risk, which is partly dependent on HDL-C levels. In addition, serum PON-1 activity provides no significant improvement in CVD risk assessment beyond conventional CVD risk factors.

Sphingosine-1-phosphate reduces ischaemia-reperfusion injury by phosphorylating the gap junction protein Connexin43.

AIM: Increasing evidence points to lipoprotein composition rather than reverse cholesterol transport in the cardioprotective properties of high-density lipoproteins (HDLs). HDL binding to receptors at the surface of cardiomyocytes activates signalling pathways promoting survival, but downstream targets are largely unknown. Here, we investigate the pathways by which the sphingosine-1-phosphate (S1P) constituent of HDL limits cell death induced by cardiac ischaemia-reperfusion (I/R). METHODS AND RESULTS: Apolipoprotein M (ApoM) transgenic (Apom-Tg) mice, in which plasma S1P is increased by 296%, and wild-type (WT) mice were subjected to in vivo I/R. Infarct size, neutrophil infiltration into the infarcted area, and serum Troponin I were less pronounced in Apom-Tg mice. In vitro experiments suggest that this cardioprotection depends on direct effects of S1P on cardiomyocytes, whereas leucocyte recruitment seems only indirectly affected. Importantly, short-term S1P treatment at the onset of reperfusion was sufficient to reduce I/R injury in isolated perfused hearts. Mechanistic in vitro and ex vivo studies revealed that 5 min of S1P treatment induced phosphorylation of the gap junction protein Connexin43 (Cx43) on Serine368 (S368), which was mediated by S1P2 and S1P3, but not by S1P1, receptors in cardiomyocytes. Finally, S1P-induced reduction of infarct size after ex vivo I/R was lost in hearts of mice with a truncated C-terminus of Cx43 (Cx43(K258/KO)) or in which the S368 is mutated to a non-phosphorylatable alanine (Cx43(S368A/S368A)). CONCLUSION: Our study reveals an important molecular pathway by which modulating the apoM/S1P axis has a therapeutic potential in the fight against I/R injury in the heart.

Proteomic and lipidomic analyses of paraoxonase defined high density lipoprotein particles: Association of paraoxonase with the anti-coagulant, protein S.

PURPOSE: Characterizing high density lipoprotein (HDL) particles and their relevance to HDL function is a major research objective. One aim is to identify functionally distinct particles. To try to limit both functional and compositional heterogeneity the present study focused on paraoxonase-1 (PON1) as a target for isolation of a minor HDL subfraction. EXPERIMENTAL DESIGN: Immunoaffinity techniques were applied to isolate PON1-containing HDL (P-HDL) and total HDL (T-HDL), which were subsequently characterized and compared. RESULTS: Analyses of the lipidomes showed significant differences between the fractions in the relative concentrations of individual lipid subspecies, notably reduced levels of unsaturated lysophosphatidylcholine (p < 0.05) in P-HDL (reflected in a significantly reduced total lysophosphatidylcholine polyunsaturated fatty acid content, p < 0.004). Significant differences were also observed for the proteomes. P-HDL was highly enriched in the anti-coagulant, vitamin K activated protein S (prot S) (p < 0.0001), and alpha2 macroglobulin (p < 0.01), compared to T-HDL. Conversely, procoagulant proteins kininogen 1 and histidine-rich glycoprotein were largely excluded from P-HDL. Immunoabsorption of PON1 from plasma significantly reduced prot S anti-coagulant activity. CONCLUSIONS AND CLINICAL RELEVANCE: The P-HDL lipidome and proteome showed significant differences from T-HDL. Enrichment in anti-coagulation proteins indicates complementary functionalities within P-HDL particles and underlines their anti-atherosclerotic potential.

CD14 as a Mediator of the Mineralocorticoid Receptor-Dependent Anti-apolipoprotein A-1 IgG Chronotropic Effect on Cardiomyocytes.

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.