Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen.

The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin's potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4'-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4'-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4'-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4'-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4'-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4'-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.

PCSK9 is minimally associated with HDL but impairs the anti-atherosclerotic HDL effects on endothelial cell activation.

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting. Our analyses showed that upon apoB-depletion, total circulating PCSK9 levels were 32% of those observed in normolipidemic plasma, and only 6% of PCSK9 in the apoB-depleted plasma, including both the mature and furin-cleaved forms, was associated with HDL. We also show human recombinant PCSK9 abolished the capacity of reconstituted HDL to reduce the formation of ROS in endothelial cells, while a PCSK9-blocking antibody enhanced the capacity of human HDL (in apoB-depleted plasma) to reduce ROS formation in endothelial cells and promote endothelial cell migration. Overall, our findings suggest that PCSK9 is only minimally associated with HDL particles, but PCSK9 in apoB-depleted plasma can affect the atheroprotective properties of HDL related to preservation of endothelial function. This study contributes to the elucidation of the pathophysiological role of plasma PCSK9 and highlights further the anti-atherosclerotic effect of PCSK9 inhibition.

Urine 8-Hydroxyguanine (8-OHG) in Patients Undergoing Surgery for Colorectal Cancer.

PURPOSE: Cellular RNA is less compact than DNA, more easily accessible to ROS and therefore could be more susceptible to oxidative damage. This study was conceived in order to analyze the RNA oxidative damage in the urine of patients undergoing operation for colorectal cancer (CRC), to compare with healthy controls, and correlate with the stage. MATERIALS AND METHODS: The study population was constituted by a group of 147 patients and a group of 128 healthy controls. Urine and blood samples were collected before the colonoscopy in all participants and 24 hours post-operatively for those who underwent surgery. Urine 8-hydroxyguanine (8-OHG) was determined as marker of RNA oxidation, and serum uric acid (UA) as antioxidant marker. RESULTS: Preoperatively, 8-OHG (ng/ml) values of CRC patients were found to be significantly higher than those of controls (p = 0.001). More specifically, stages II/III had significantly higher 8-OHG values (p < 0.001 and p = 0.007) than stages 0/I. Post-operatively, 8-OHG values were similar to controls (p = 0.053). Preoperatively, UA values (mg/dl) were significantly lower (p = 0.001), while postoperatively were similar to controls (p = 0.069). CONCLUSION: Oxidative RNA damage occurs in CRC patients. Stages II/III are associated with higher values of 8-OHG than stages 0/I. 8-OHG could act as a marker for the identification of patients with advanced disease.

Pharmacology of PCSK9 Inhibitors: Current Status and Future Perspectives.

Protein Convertase Subtilisin/Kexin type 9 (PCSK9) is a serine protease primarily expressed in the liver, which represents the main source of the plasma enzyme. The best characterized function of PCSK9 relates to the binding to Low-Density Lipoprotein Receptor (LDL-R) in hepatocytes, increasing its endosomal and lysosomal degradation. This results in the inhibition of LDL-R recycling to the cell surface and therefore the reduction of the hepatic uptake of LDL, leading to the increase in plasma levels of LDL-cholesterol, a major risk factor of Cardiovascular Diseases (CVD). Therefore, PCSK9 is an important therapeutic target to reduce LDLcholesterol levels. PCSK9 inhibition can occur at the level of its interaction with LDL-R as well as at several sites across the pathway of its intracellular synthesis and secretion. Two fully human mAbs, Alirocumab and Evolocumab, that selectively bind to PCSK9 and prevent its interaction with the LDL-R, are currently used in the clinical practice. These mAbs are the most potent cholesterol-lowering agents available today and can decrease LDLcholesterol levels up to 73% while they also reduce the risk of atherosclerotic CVD. Ongoing research has led to the development of new PCSK9 inhibitors through genome editing technology (CRISPR-Cas9), siRNA or antisense oligonucleotide silencing agents, vaccines, mimetic peptides, adnectins, and inhibitors of PCSK9 secretion. The above inhibitors have been studied in vitro, in animal models in vivo, as well as in phase I and II trials and have demonstrated an important efficacy profile. Future studies with these agents will demonstrate their possible clinical value and will further enlighten the various targets and activities of PCSK9 intracellularly and extracellularly, the underlying mechanisms, as well as the clinical significance of these actions beyond the inhibition of LDL-R recycling.

Effects of increased body weight and short-term weight loss on serum PCSK9 levels - a prospective pilot study.

INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with low-density lipoprotein (LDL) catabolism, but its serum concentration is not uniformly associated with cardiovascular disease in clinical studies. Obesity is linked with increased cardiovascular risk, but the effect of increased body weight and short-term weight loss on serum PCSK9 levels is not well studied. MATERIAL AND METHODS: The aim of this prospective pilot study was to assess differences in serum PCSK9 levels (determined with a quantitative sandwich enzyme immunoassay) between otherwise healthy drug-naïve obese subjects and healthy individuals with normal body weight. Additionally, PCSK9 levels were determined at baseline and after a 3-month weight-loss program with a low-fat diet in a randomly assigned subgroup of the obese subjects (n = 15). RESULTS: Obese subjects (n = 35) were older (age: 43 ±11 years) and had significantly higher body mass index, total cholesterol, triglycerides, LDL cholesterol (LDL-C), apolipoprotein B and homeostasis model assessment of insulin resistance (HOMA) index levels, as well as significantly lower high-density lipoprotein cholesterol (HDL-C) concentration, compared with normal-weight subjects (n = 20, age: 35 ±6 years). Serum PCSK9 levels were significantly higher in obese subjects compared with normal-weight individuals, even after adjustment for age, LDL-C, triglycerides, HDL-C, apolipoprotein A1, apolipoprotein B, apolipoprotein E, glucose, insulin and HOMA index levels (p = 0.018). Obese subjects experienced significant weight loss (from 109 ±22 to 104 ±23 kg, p < 0.01), but serum PCSK9 levels did not significantly change after the 3-month weight-loss program. CONCLUSIONS: Serum PCSK9 levels are higher in obese subjects than in normal-weight individuals. Short-term weight loss with a low-fat diet does not significantly affect PCSK9 levels.

Pleiotropic effects of apolipoprotein C3 on HDL functionality and adipose tissue metabolic activity.

APOC3 is produced mainly by the liver and intestine and approximately half of plasma APOC3 associates with HDL. Though it was believed that APOC3 associates with HDL by simple binding to preexisting particles, recent data support that biogenesis of APOC3-containing HDL (APOC3-HDL) requires Abca1. Moreover, APOC3-HDL contributes to plasma triglyceride homeostasis by preventing APOC3 association with triglyceride-rich lipoproteins. Interestingly, APOC3-HDL also shows positive correlation with the morbidly obese phenotype. However, the roles of APOC3 in HDL functionality and adipose tissue metabolic activity remain unknown. Therefore, here we investigated the direct effects of APOC3 expression on HDL structure and function, as well as white adipose tissue (WAT) and brown adipose tissue (BAT) metabolic activity. C57BL/6 mice were infected with an adenovirus expressing human APOC3 or a recombinant attenuated control adenovirus expressing green fluorescent protein and blood and tissue samples were collected at 5 days postinfection. HDL was then analyzed for its apolipoprotein and lipid composition and particle functionality. Additionally, purified mitochondria from BAT and WAT were analyzed for uncoupling protein 1, cytochrome c (Cytc), and Cytc oxidase subunit 4 protein levels as an indirect measure of their metabolic activity. Serum metabolomic analysis was performed by NMR. Combined, our data show that APOC3 modulates HDL structure and function, while it selectively promotes BAT metabolic activation.

Hypertrophic Mesenteric Adipose Tissue May Play a Role in Atherogenesis in Inflammatory Bowel Diseases.

BACKGROUND: Adipokines released by the adipose tissue are known to play a role in atherogenesis. The hypertrophic mesenteric fat in patients with inflammatory bowel diseases (IBD) also produces adipokines that are considered to play a role in intestinal inflammation. Whether they also contribute to accelerated atherosclerosis in IBD is unknown. The aim of this study was to assess the role of 2 adipokines, resistin and adiponectin, in IBD. METHODS: We previously published data on 3 markers of cardiovascular risk, carotid intima-media thickness, carotid-femoral pulse wave velocity, and lipoprotein-associated phospholipase A2, in 44 patients with IBD and 44 controls matched for established cardiovascular risk factors. In this study, we measured resistin and adiponectin levels, and assessed their correlations with carotid intima-media thickness, pulse wave velocity, and lipoprotein-associated phospholipase A2. RESULTS: Resistin levels were significantly higher in patients with IBD (13.7 versus 10 ng/mL; P = 0.022), but there was no difference in adiponectin levels. Resistin levels were significantly higher in patients with active disease compared with those in remission (18.9 versus 11.3 ng/mL; P = 0.014). Adiponectin levels were significantly lower in Crohn's disease compared with ulcerative colitis (6736.3 ± 3105 versus 10,476.1 ± 5575.7 ng/mL; P = 0.026). Adiponectin correlated inversely with pulse wave velocity (rho = -0.434; P < 0.0005) and carotid intima-media thickness (rho = -0.255; P = 0.021). CONCLUSIONS: This is the first study to suggest that adipokines produced by the hypertrophic mesenteric fat in IBD may play a role not only in intestinal inflammation but also in atherogenesis. Resistin has mainly pro-inflammatory properties, whereas adiponectin likely exerts an angioprotective effect.

The relationship between retinol-binding protein 4 and apolipoprotein B-containing lipoproteins is attenuated in patients with very high serum triglycerides: A pilot study.

OBJECTIVE: The investigation of the association between retinol-binding protein 4 (RBP4) and lipoproteins in subjects with hypertriglyceridemia. DESIGN: Forty-six obese or overweight hypertriglyceridemic patients were studied at baseline and 20 of them underwent a hypocaloric low-fat diet for 3 months. RESULTS: Plasma RBP4 levels were positively correlated with serum triglycerides (TG) in the subgroup of patients with TG <200 mg/dL (r=0.453, p=0.039) and negatively correlated with TG in patients with TG ≥200 mg/dL (r=-0.487, p=0.019). In the subgroup with TG <200 mg/ dL, subjects with circulating RBP4 above the median 46 mg/L had higher levels of intermediate density lipoprotein-cholesterol (IDL-C), low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (ApoB), while these differences were absent in patients with TG ≥200 mg/dL. The associations of percentage changes of circulating RBP4 with the percentage changes of LDL-C, very low-density lipoprotein-cholesterol (VLDL-C) and ApoB were positive after the first month and 3 months of diet for patients with baseline TG <200 mg/dL, while no correlations existed for patients with TG ≥200 mg/dL. CONCLUSIONS: The positive association between circulating RBP4 and ApoB-containing lipoproteins in a steady metabolic state, as well as during a hypocaloric diet, appears to be attenuated in patients with very high TG.

Amyloid-beta (1-40) and the risk of death from cardiovascular causes in patients with coronary heart disease.

BACKGROUND: The amyloid beta peptide is the major protein constituent of neuritic plaques in Alzheimer disease and appears to play a central role in vascular inflammation pathophysiology. OBJECTIVES: This study sought to determine the clinical value of amyloid-beta 1-40 (Abeta40) measurement in predicting cardiovascular (CV) mortality in patients with coronary heart disease (CHD) and arterial stiffness progression in young healthy subjects. METHODS: Abeta40 was retrospectively measured in blood samples collected from 3 independent prospective cohorts and 2 case-control cohorts (total N = 1,464). Major adverse cardiac events (MACE) were assessed in the 2 prospective cohorts (n = 877) followed for a median of 4.4 years. To look at effects on subclinical disease, arterial stiffness was evaluated at baseline and after 5-year follow-up (n = 107) in young healthy subjects. The primary endpoint was the predictive value of Abeta40 for CV mortality and outcomes in patients with CHD. RESULTS: In Cox proportional hazards models adjusted for age, sex, estimated glomerular filtration rate, left ventricular ejection fraction, high-sensitivity C-reactive protein, and high-sensitivity troponin T, Abeta40 independently predicted CV death and MACE in patients with CHD (p < 0.05 for all). After multivariate adjustment, Abeta40 levels conferred a substantial enhancement of net reclassification index and integrated discrimination improvement of individuals at risk in the total combined CHD cohort over the best predictive model. Further cohort-based analysis revealed that Abeta40 levels were significantly and independently associated with arterial stiffness progression, incident subclinical atherosclerosis, and incident CHD. CONCLUSIONS: Measuring blood levels of Abeta40 identified patients at high risk for CV death.

Oxidative stress in multi-port and single-port cholecystectomy.

BACKGROUND: This study was designed to analyze and compare plasma levels of 8-isoprostane (8-epiPGF2α), a biomarker of lipid peroxidation, and uric acid (UA), a marker of the antioxidant status, in standard laparoscopic (LC) and laparoendoscopic single-site cholecystectomy (LSSC). MATERIALS AND METHODS: Forty patients with noncomplicated cholelithiasis were randomized to undergo either LSSC (n = 20) or LC (n = 20). The patients had body mass index <30, American Society of Anesthesiologists score I or II, and no previous upper gastrointestinal surgery. Blood samples were taken preoperatively and 6 h and 24 h postoperatively. Levels of 8-epiPGF2α were determined using enzyme-linked immunosorbent assay, whereas levels of UA were calculated using automated analyzer. RESULTS: No significant differences were observed in operative data among the groups. Levels of 8-epiPGF2α were significantly higher in LSSC compared with LC at 6 h (P = 0.003) and 24 h (P < 0.001). 8-epiPGF2α levels showed significant changes over time in LC (LSSC: P = 0.720, LC: P < 0.001). UA levels were significantly higher in LC compared with LSSC, 24 h postoperatively (P = 0.021). No significant changes over time in the UA levels in both groups (LSSC: P = 0.056, LC: P = 0.205). CONCLUSIONS: LSSC is associated with increased oxidative stress compared with LC. Further studies are needed to confirm these results.

Pathophysiological role and clinical significance of lipoprotein-associated phospholipase A2 (Lp-PLA2) bound to LDL and HDL.

Lipoprotein-associated phospholipase A2 (Lp-PLA2), also named as platelet-activating factor (PAF)-acetylhydrolase, exhibits a Ca2+-independent phospholipase A2 activity and catalyzes the hydrolysis of the ester bond at the sn-2 position of PAF and oxidized phospholipids (oxPL). These phospholipids are formed under oxidative and inflammatory conditions, and may play important roles in atherogenesis. The vast majority of plasma Lp-PLA2 mass binds to low-density lipoprotein (LDL) while a smaller amount is associated with high-density lipoprotein (HDL). Lp-PLA2 is also bound to lipoprotein (a) [Lp(a)], very low-density lipoprotein (VLDL) and remnant lipoproteins. Several lines of evidence suggest that the role of plasma Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. Data from large Caucasian population studies have supported plasma Lp-PLA2 (primarily LDL-associated Lp-PLA2) as a cardiovascular risk marker independent of, and additive to, traditional risk factors. On the contrary, the HDL-associated Lp-PLA2 may express antiatherogenic activities and is also independently associated with lower risk for cardiac death. The present review presents data on the biochemical and enzymatic properties of Lp-PLA2 as well as its structural characteristics that determine the association with LDL and HDL. We also critically discuss the possible pathophysiological and clinical significance of the Lp- PLA2 distribution between LDL and HDL in human plasma, in view of the results of prospective epidemiologic studies on the association of Lp-PLA2 with future cardiovascular events as well the recent studies that evaluate the possible effectiveness of specific Lp-PLA2 inhibitors in reducing residual cardiovascular risk.

Lipoprotein-associated phospholipase A2 and arterial stiffness evaluation in patients with inflammatory bowel diseases.

BACKGROUND AND AIMS: The association between inflammatory bowel diseases (IBD) and cardiovascular disease (CVD) remains equivocal. Arterial stiffness, as assessed by pulse wave velocity (PWV), and lipoprotein-associated phospholipase A2 (Lp-PLA2) are surrogates of CVD risk. AIM: The aim of this study was to assess carotid-femoral PWV and Lp-PLA2 in patients with IBD without history of CVD. METHODS: Established CVD risk factors, IBD characteristics, PWV and Lp-PLA2 activity were assessed in 44 patients with IBD, 29 with Crohn's disease (CD) and 15 with ulcerative colitis (UC), and 44 matched controls. RESULTS: IBD patients had lower total and low density lipoprotein cholesterol (LDL-C) levels. There was no difference in PWV between patients and controls (6.8 vs. 6.4m/s), but patients with CD had higher PWV compared to those with UC (7 vs. 6.3m/s; p=0.044), and to controls. Smoking rates were significantly higher among CD patients. Factors associated with PWV were age, mean arterial pressure and smoking. Lp-PLA2 activity was significantly lower in patients with IBD (46.8 vs. 53.9 nmol/mL/min; p=0.011). There was no difference in Lp-PLA2 between CD and UC patients. LDL-C was the only significant predictor of Lp-PLA2. CONCLUSIONS: Our study showed lower Lp-PLA2 activity in patients with IBD compared with controls, reflecting lower LDL-C in the former. There was no difference in PWV between the two groups. Arterial stiffness was higher in patients with CD, which is likely related to higher smoking rates. These findings challenge a possible association between IBD and CVD, but further studies are required.

Differential membrane expression of Toll-like receptors and intracellular cytokine induction in peripheral blood monocytes of patients with chronic kidney disease and diabetic nephropathy.

BACKGROUND: Toll-like receptors (TLRs) are key players in the innate immune system whose activation leads to an inflammatory response. Inflammation plays an important role in the pathogenesis of chronic kidney disease (CKD) and diabetes mellitus. The aim of our study was to assess the proinflammatory state of nondialysis CKD patients by evaluating the membrane expression of TLR2 and TLR4 and the intracellular IL-1ß and IL-6 production in response to the ligand Pam3Cys-Ser-(Lys)4 (Pam3CSK4). METHODS: 85 nondialysis CKD patients [mean estimated glomerular filtration rate: 34 (17-90) ml/min/1.73 m(2)] were divided in 2 groups: 55 nondiabetic CKD patients (CKD group) and 30 patients with diabetic nephropathy (DN group). The two groups were compared with 36 healthy subjects (control group). TLR2 and TLR4 membrane expression in monocytes and Pam3CSK4-induced intracellular production of IL-1ß and IL-6 were assessed by flow cytometry. RESULTS: Both patient groups showed increased TLR2 membrane expression compared with the control group, both at baseline (p < 0.05 for both) and after Pam3CSK4 stimulation (p < 0.05 for both). The DN group exhibited significantly higher TLR4 expression at baseline compared to the CKD and control groups (p < 0.04 and p < 0.02, respectively). Intracellular IL-1ß and IL-6 levels at baseline were significantly lower in CKD patients compared to the DN and control groups. After Pam3CSK4 stimulation, intracellular IL-1ß and IL-6 increased in all groups, but were lower in the CKD group versus the control group or DN group, which exhibited higher levels than the controls. CONCLUSIONS: Nondialysis CKD patients showed significant alterations in TLR2 and TLR4 membrane expression, and impaired Pam3CSK4-induced cytokine production in monocytes, a phenomenon that is markedly influenced by the presence of diabetes.

Comparison of the effect of simvastatin versus simvastatin/ezetimibe versus rosuvastatin on markers of inflammation and oxidative stress in subjects with hypercholesterolemia.

OBJECTIVES: Statins may exhibit anti-inflammatory and antioxidant effects. Whether different statins at equivalent doses or the combination of low-dose statin with ezetimibe have comparable anti-inflammatory and antioxidant effects is unknown. The aim of this study was to compare the effects of simvastatin, simvastatin/ezetimibe or rosuvastatin at equivalent low-density lipoprotein cholesterol lowering doses on inflammation and oxidative stress indices in subjects with hypercholesterolemia. METHODS: This was a pre-specified analysis of a prospective, randomized, open-label, blinded endpoint (PROBE) study. We enrolled one hundred and fifty three (n = 153) hypercholesterolemic subjects who were randomized to receive simvastatin 40 mg or simvastatin/ezetimibe 10/10 mg or rosuvastatin 10 mg daily. Plasma 8-Epi prostaglandin F2 alpha (8-epiPGF2a), oxidized LDL (oxLDL) and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and mass were measured at baseline and following 12 weeks of treatment. RESULTS: A significant reduction in plasma 8-isoprostane and oxLDL levels was observed in all treatment groups [by 10%, 8% and 6% (p < 0.05 compared with baseline) and 41%, 40% and 39% (p < 0.001 compared with baseline) in simvastatin, simvastatin/ezetimibe and rosuvastatin groups, respectively]. In all treatment groups a significant reduction in total plasma Lp-PLA2 activity and mass was observed (by 36%, 31% and 38% and 36%, 32% and 32% for simvastatin, simvastatin/ezetimibe and rosuvastatin, respectively, p < 0.001 compared with baseline). No intergroup differences were observed. CONCLUSIONS: Simvastatin 40 mg, simvastatin/ezetimibe 10/10 mg and rosuvastatin 10 mg significantly reduced 8-epiPGF2a, oxLDL and Lp-PLA2 activity and mass to a similar extent.

The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2⁻] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2⁻ were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2⁻ levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2⁻. Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.

The effect of combining rosuvastatin with sartans of different peroxisome proliferator receptor-γ activating capacity on plasma 8-isoprostane prostaglandin F2a levels.

INTRODUCTION: Oxidative stress is associated with the development and progression of cardiovascular disease. Plasma 8-isoprostane prostaglandin F2a (8-iso-PGF2a) levels are a reliable marker of oxidative stress. MATERIAL AND METHODS: Patients (n = 151) with hypertension, dyslipidemia and impaired fasting glucose were randomly allocated to rosuvastatin (10 mg/day) plus telmisartan 80 mg/day (RT group, n = 52) or irbesartan 300 mg/day (RI group, n = 48) or olmesartan 20 mg/day (RO group, n = 51). After 6 months of treatment, changes in plasma 8-iso-PGF2a levels were blindly evaluated. RESULTS: A decrease of 8-iso-PGF2a levels vs baseline was observed only in the RT group (-8.6%; p = 0.02). A trend for decrease vs. baseline was observed in the RI (-5.7%; p = 0.40) and RO (-3.7%; p = 0.60) groups. Changes of 8-iso-PGF2a levels between groups were not significantly different (p = 0.70). CONCLUSIONS: The combination of rosuvastatin with sartans of different peroxisome proliferator receptor-γ activating capacity was associated with a decrease in levels of plasma 8-iso-PGF2a. This decrease reached significance only in the telmisartan group.

Lipoprotein-associated phospholipase A(2) bound on high-density lipoprotein is associated with lower risk for cardiac death in stable coronary artery disease patients: a 3-year follow-up.

OBJECTIVES: The aim of this study was to examine the prognostic value of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) associated with high-density lipoprotein (HDL) (HDL-Lp-PLA(2)) in patients with stable coronary artery disease (CAD). BACKGROUND: Lp-PLA(2) is a novel risk factor for cardiovascular disease. It has been postulated that the role of Lp-PLA(2) in atherosclerosis may depend on the type of lipoprotein with which it is associated. METHODS: Total plasma Lp-PLA(2) and HDL-Lp-PLA(2) mass and activity, lipids, and C-reactive protein were measured in 524 consecutive patients with stable CAD who were followed for a median of 34 months. The primary endpoint was cardiac death, and the secondary endpoint was hospitalization for acute coronary syndromes, myocardial revascularization, arrhythmic event, or stroke. RESULTS: Follow-up data were obtained from 477 patients. One hundred twenty-three patients (25.8%) presented with cardiovascular events (24 cardiac deaths, 47 acute coronary syndromes, 28 revascularizations, 22 arrhythmic events, and 2 strokes). Total plasma Lp-PLA(2) mass and activity were predictors of cardiac death (hazard ratio [HR]: 1.013; 95% confidence interval [CI]: 1.005 to 1.021; p = 0.002; and HR: 1.040; 95% CI: 1.005 to 1.076; p = 0.025, respectively) after adjustment for traditional risk factors for CAD. In contrast, HDL-Lp-PLA(2) mass and activity were associated with lower risk for cardiac death (HR: 0.972; 95% CI: 0.952 to 0.993; p = 0.010; and HR: 0.689; 95% CI: 0.496 to 0.957; p = 0.026, respectively) after adjustment for traditional risk factors for CAD. CONCLUSIONS: Total plasma Lp-PLA(2) is a predictor of cardiac death, while HDL-Lp-PLA(2) is associated with lower risk for cardiac death in patients with stable CAD, independently of other traditional cardiovascular risk factors.

Effect of simvastatin or its combination with ezetimibe on Toll-like receptor expression and lipopolysaccharide - induced cytokine production in monocytes of hypercholesterolemic patients.

OBJECTIVES: Toll-like receptors (TLRs) are key players in the innate immune system. Recently, a pivotal role of TLR2 and TLR4 has been recognized in atherogenesis. We investigated the effect of simvastatin monotherapy or its combination with ezetimibe on TLR2 and TLR4 membrane expression and on lipopolysaccharide (LPS)-induced interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) production in peripheral blood monocytes of patients with primary hypercholesterolemia. METHODS: This was a prospective, randomized, open-label, blinded endpoint study. After a 3-month period of lifestyle changes patients (n = 60) (mean age 55 ± 13) with LDL-cholesterol levels above those recommended by the NCEP ATP III, were randomly allocated to open-label simvastatin 40 mg (n = 30) or simvastatin/ezetimibe 10/10 mg (n = 30) daily. Both groups were similar with regard to demographics, risk factors, medications and baseline lipid values. TLR2 and TLR4 membrane expression in monocytes, LPS-induced intracellular production of IL-1ß and IL-6 were assessed by flow cytometry at baseline and 3 months post-treatment in both patient groups, as well as in 30 age- and sex-matched normolipidemic controls. RESULTS: Hypercholesterolemic patients exhibited higher TLR2 and TLR4 membrane expression compared with controls (p < 0.02). LPS induced a significant increase in the intracellular levels of IL-1ß and IL-6 in all groups however both patient groups exhibited significantly lower levels compared with controls. Three months of treatment with either simvastatin or its combination with ezetimibe resulted in a significant reduction of TLR2 and TLR4 expression (p < 0.01 compared with baseline values) with no intergroup differences. Furthermore, in both groups the post-treatment values of LPS-induced IL-1ß and IL-6 production were significantly lower compared with baseline (p < 0.05 for all comparisons). CONCLUSIONS: A high simvastatin dose or the combination of a low-dose simvastatin with ezetimibe reduce to a similar extent TLR2, TLR4 membrane expression and LPS-induced IL-6 and IL-1ß production in monocytes of hypercholesterolemic patients. The pathophysiological significance of these effects regarding atherosclerosis, reserves further investigation.

Plasma Lp-PLA(2) mass and apoB-lipoproteins that carry Lp-PLA(2) decrease after sodium.

BACKGROUND: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) ) is a novel cardiovascular risk marker, which is predominantly complexed to apolipoprotein (apo) B-containing lipoproteins in human plasma. As increasing dietary sodium intake may decrease plasma apoB-containing lipoproteins, we tested whether a sodium challenge lowers plasma Lp-PLA(2) mass, as well as the levels of apoB-containing lipoprotein particles carrying Lp-PLA(2) (apoB-Lp-PLA(2) ), employing a newly developed enzyme-linked immunosorbent assay. MATERIALS AND METHODS: In 45 women and 31 men (mean age 44 ± 14 years), plasma Lp-PLA(2) mass (turbidimetric immunoassay), the level of apoB-Lp-PLA(2) , expressed in apoB concentration and lipoproteins were measured in response to a 3-day challenge with 9 g sodium chloride tablets daily. RESULTS: Urinary sodium excretion increased from 165 ± 60 to 321 ± 70 mmol/24 h (P<0.001) after salt loading. Plasma Lp-PLA(2) mass decreased from 618 (493-719) to 588 (465-698) µg/L (P<0.001), and apoB-Lp-PLA(2) decreased from 0.276 (0.200-0.351) to 0.256 (0.189-0.328) g LDL protein/L (P=0.004) in response to the sodium challenge together with decreases in plasma total cholesterol, nonhigh-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and the total cholesterol/HDL cholesterol ratio (P<0.01 for all). Changes in plasma Lp-PLA(2) mass were correlated positively with changes in total cholesterol, LDL cholesterol and non-HDL cholesterol (r=0.260-0.276, P<0.05 to P<0.02), whereas changes in apoB-Lp-PLA(2) were correlated positively with changes in non-HDL cholesterol and in the total cholesterol/HDL cholesterol ratio (r=0.232-0.385, P<0.05-0.01). CONCLUSION: Both plasma Lp-PLA(2) mass levels and apoB-Lp-PLA(2) decrease in response to a short-term oral sodium challenge.

The changes in plasma retinol-binding protein 4 levels are associated with those of the apolipoprotein B-containing lipoproteins during dietary and drug treatment.

We investigated the association between retinol-binding protein 4 (RBP(4)) and apolipoprotein B (ApoB)-containing lipoproteins. Obese or overweight, hypertriglyceridemic patients underwent the following interventions for 3 months: (1) Diet (n = 20), (2) Diet + fenofibrate (n = 18), (3) Diet + rimonabant (n = 8). Circulating RBP4 decreased during dietary treatment. The percentage change in RBP(4) was positively correlated with the percentage changes in very-low density lipoprotein cholesterol (r = .570, P = .02), low-density lipoprotein cholesterol ([LDL-C]; r = .605, P = .01), ApoB (r = .705, P = .007), and small dense LDL-C ([sdLDL-C]; r = .872, P < .001). The percentage change in RBP4 was the best predictor of the percentage changes in sdLDL-C and ApoB. Rimonabant treatment reduced RBP4, whereas fenofibrate increased RBP4 during the first month of therapy followed by a subsequent decrease. In conclusion, RBP4 may significantly influence the metabolic pathways responsible for changes in ApoB lipoprotein subspecies, thus RBP4 may be associated with cardiovascular disease risk.