Both paraoxonase-1 genotype and activity do not predict the risk of future coronary artery disease; the EPIC-Norfolk Prospective Population Study.

BACKGROUND: Paraoxonase-1 (PON1) is an antioxidant enzyme, that resides on high-density lipoprotein (HDL). PON1-activity, is heavily influenced by the PON1-Q192R polymorphism. PON1 is considered to protect against atherosclerosis, but it is unclear whether this relation is independent of its carrier, HDL. In order to evaluate the atheroprotective potential of PON1, we assessed the relationships among PON1-genotype, PON1-activity and risk of future coronary artery disease (CAD), in a large prospective case-control study. METHODOLOGY/PRINCIPAL FINDINGS: Cases (n = 1138) were apparently healthy men and women aged 45-79 years who developed fatal or nonfatal CAD during a mean follow-up of 6 years. Controls (n = 2237) were matched by age, sex and enrollment time. PON1-activity was similar in cases and controls (60.7+/-45.3 versus 62.6+/-45.8 U/L, p = 0.3) and correlated with HDL-cholesterol levels (r = 0.16, p<0.0001). The PON1-Q192R polymorphism had a profound impact on PON1-activity, but did not predict CAD risk (Odds Ratio [OR] per R allele 0.98[0.84-1.15], p = 0.8). Using conditional logistic regression, quartiles of PON1-activity showed a modest inverse relation with CAD risk (OR for the highest versus the lowest quartile 0.77[0.63-0.95], p = 0.01; p-trend = 0.06). PON1-activity adjusted for Q192R polymorphism correlated better with HDL-cholesterol (r = 0.26, p<0.0001) and more linearly predicted CAD risk (0.79[0.64-0.98], p = 0.03; p-trend = 0.008). However, these relationships were abolished after adjustment for HDL (particles-cholesterol-size) and apolipoproteinA-I (0.94[0.74-1.18], p-trend = 0.3). CONCLUSIONS/SIGNIFICANCE: This study, shows that PON1-activity inversely relates to CAD risk, but not independent of HDL, due to its close association with the HDL-particle. These data strongly suggest that a low PON1-activity is not a causal factor in atherogenesis.

C-reactive protein elicits white blood cell activation in humans.

OBJECTIVE: Consistent epidemiologic evidence suggests that acute infections increase the risk for acute cardiovascular events. We tested in humans whether activation of peripheral leukocytes in reaction to the administration of recombinant human C-reactive protein (rhCRP) may provide a mechanism for infectious diseases to promote atherosclerotic disease. METHODS AND RESULTS: By using quantitative real-time polymerase chain reaction analysis, whole-blood expression profiles were analyzed for 95 inflammatory markers before and after infusion of 1.25 mg/kg rhCRP in 5 male volunteers. Relevant transcript levels were measured at baseline and 4 and 8 hours after rhCRP-infusion. CRP caused significant up-regulation of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-1, plasminogen activator urokinase, macrophage inflammatory protein 1 alpha, and nuclear factor of kappa B inhibitor mRNAs in peripheral leukocytes. mRNA up-regulation of MMP-9 and MCP-1 was 17- and 11-fold, respectively. The corresponding increase in plasma protein levels of MMP-9 (78+/-32 ng/mL to 109+/-41 ng/mL; P=.014) and MCP-1 (312+/-92 pg/mL to 2590+/-898 pg/mL; P=.007) closely mirrored mRNA findings. Also, in whole-blood culture stimulation assays, CRP induced proinflammatory changes. Notably, heat inactivation abolished the capacity of CRP to evoke these proinflammatory changes, excluding a role for contaminants within the purified CRP preparation. CONCLUSION: CRP elicits activation of peripheral leukocytes with ensuing secretion of plaque-destabilizing mediators. These findings are consistent with the hypothesis that infectious diseases trigger manifestations of atherosclerosis, in which CRP elevation might contribute to the onset of cardiovascular events.

Effects of short-term and long-term danazol treatment on lipoproteins, coagulation, and progression of atherosclerosis: two clinical trials in healthy volunteers and patients with hereditary angioedema.

BACKGROUND: Danazol is a synthetic androgen derivative frequently used as prophylaxis in patients with hereditary angioedema (HAE) due to complement-1 esterase inhibitor deficiency. However, danazol has been reported to decrease high-density lipoprotein cholesterol (HDL-C) levels and to adversely affect coagulation parameters, which are considered to be proatherothrombotic. OBJECTIVE: The short- and long-term effects of danazol were evaluated on proatherogenic intermediate end points in healthy volunteers and patients with HAE. METHODS: Short-term effects were evaluated in healthy men randomly assigned to 200 mg/d of danazol or placebo for 4 weeks in a crossover trial with no washout period. Long-term effects of danazol on lipoproteins, coagulation, and carotid intima-media thickness (CIMT) were evaluated in a cross-sectional study in which patients with HAE treated with danazol, a mean dose of 170 mg/d for >or=2 years, were compared with healthy controls matched for age, sex, and body mass index (BMI). Drug tolerability was assessed by questionnaires and adherence was measured by pill count when drug bottles were returned after every study visit. RESULTS: Patients in the short-term study were 15 men with a mean (SD) age of 32.6 (6.9) years and BMI of 24.3 (4.1) kg/m(2). In the long-term study, patients with HAE were 10 women and 7 men with a mean (SD) age of 41.1 (12.9) years and BMI of 25.4 (2.6) kg/m(2); the 17 matched controls had a mean (SD) age of 39.8 (11.8) years and BMI of 25.4 (2.6) kg/m(2). Short-term danazol treatment was associated with a decrease from baseline in apolipoprotein A-I of 21% and in HDL-C of 23%. Flow-mediated dilation and coagulation parameters were unaffected after 4 weeks. Longterm danazol treatment did not adversely affect HDL-C concentration (1.1 [0.5] vs baseline, 1.2 [0.5] pmol/L), HDL-related transfer proteins such as paraoxonase-1 activity (92 [62] vs 80 [40] U/mM), cholesteryl-ester transfer protein mass (1.5 [0.4] vs 2.2 [0.6] microg/mL), lecithin cholesterol acyltransferase activity (21.2 [4.5] vs 32.1 [7.2] nmol CE . mL(-1) . h(-1)), plasma phospholipid transfer protein activity (15.4 [1.5] vs 14.9 [1.2] AU), and apolipoproteins between patients with HAE and controls. The mean (SD) CIMT was similar between patients with HAE and controls (0.62 [0.09] vs 0.59 [0.08] mm; P = NS). However, HAE patients using danazol had increased coagulation activation when compared with controls (prothrombin fragments, 286 [119] vs 164 [57] pmol/L, P = 0.002; thrombinantithrombin complex, 3.9 [1.4] vs 2.6 [1.1] microg/L, P = 0.01). CONCLUSIONS: Short-term danazol treatment in healthy volunteers was associated with a reduction in HDL-C levels without a significant effect on endothelial function or coagulation parameters. In contrast, patients with HAE treated for >2 years with danazol had increased activation of coagulation, but there were no significant differences in HDL-C or CIMT compared with matched healthy controls.

Induction of IRAK-M is associated with lipopolysaccharide tolerance in a human endotoxemia model.

Recent in vitro and murine in vivo studies have identified several potential LPS tolerance factors. In this study, we describe the expression kinetics of these LPS tolerance factors in standardized human endotoxemia models using i.v. LPS bolus administration. Responsiveness to LPS as well as the expression of potential regulators of LPS signaling were determined in peripheral whole blood. Intravenous LPS administration (4 ng/kg) resulted in peak plasma levels of TNF-alpha at 1.5 h followed by subsequent peaks of the classic negative feedback inhibitors A20 and IL-10 at 2 and 3 h, respectively. Circulating blood monocyte counts decimated during the initial inflammatory response, but normalized in the period between 4 and 8 h post-LPS. The LPS response as determined by ex vivo TNF release per monocyte in whole blood was profoundly decreased at 6-8 h post-LPS injection despite cessation of A20 and IL-10 expression after 4 h. Analysis of MyD88short, IL-1R-associated kinase (IRAK)-1, IRAK-M, ST2, suppressor of cytokine signaling-1 and -3, SHIP-1, and MAP kinase phosphatase-1 expression indicated that the observed LPS tolerance was associated with decreased IRAK-1 and elevated IRAK-M expression in this human model. Interestingly, a lower dose of LPS (1 ng/kg) induced LPS tolerance accompanied with IRAK-M up-regulation but without depletion of IRAK-1. In vitro studies in whole blood showed that IRAK-M up-regulation by LPS is largely dependent on TNF-alpha. The observed rise of IRAK-M transcription in the human endotoxemia model appeared much greater compared with in vitro-stimulated whole blood. In conclusion, LPS tolerance in human endotoxemia models is associated with IRAK-M up-regulation.

Apolipoprotein A-II is inversely associated with risk of future coronary artery disease.

BACKGROUND: Although the vasculoprotective effects of apolipoprotein A-I (apoA-I), the major protein associated with high-density lipoprotein, have been universally accepted, apoA-II has been suggested to have poor antiatherogenic or even proatherogenic properties. To study this suggestion more closely, we evaluated how serum levels of apoA-II and apoA-I relate to the risk of future coronary artery disease (CAD) in a large, prospective study. METHODS AND RESULTS: We performed a nested case-control study in the prospective EPIC-Norfolk (European Prospective Investigation into Cancer and Nutrition-Norfolk) cohort. Case subjects (n=912) were apparently healthy men and women aged 45 to 79 years who developed fatal or nonfatal CAD during a mean follow-up of 6 years. Control subjects (n=1635) were matched by age, gender, and enrollment time. Conditional logistic regression was used to quantify the relationship between serum apoA-II levels and risk of CAD. Serum apoA-II concentration was significantly lower in case subjects (34.5+/-6.3 mg/dL) than in control subjects (35.2+/-5.8 mg/dL) and was inversely associated with risk of CAD, such that patients in the upper quartile (>38.1 mg/dL) had an odds ratio of 0.59 (95% confidence interval 0.46 to 0.76) versus those in the lowest quartile (<31.1 mg/dL; P for linearity <0.0001). After adjustment for fasting time, alcohol use, and cardiovascular risk factors (systolic blood pressure, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, body mass index, smoking, diabetes mellitus, and C-reactive protein), the corresponding risk estimate was 0.48 (95% confidence interval 0.34 to 0.67, P for linearity <0.0001). Surprisingly, additional adjustment for serum apoA-I levels did not affect risk prediction of apoA-II for future CAD (odds ratio 0.49, 95% confidence interval 0.34 to 0.68, P for linearity <0.0001). Also, after adjustment for high-density lipoprotein particle number and size, apoA-II was still associated with the risk of future CAD (odds ratio 0.62, 95% confidence interval 0.43 to 0.90, P for linearity 0.02). CONCLUSIONS: ApoA-II is associated with a decreased risk of future CAD in apparently healthy people. These findings imply that apoA-II itself exerts effects on specific antiatherogenic pathways. On the basis of these findings, discussion of the potential proatherogenic effects of apoA-II can cease.

A single bolus infusion of C-reactive protein increases gluconeogenesis and plasma glucose concentration in humans.

Recently, we reported that C-reactive protein (CRP) elicits inflammatory and procoagulant responses in humans. In addition, CRP has been associated with the development of type 2 diabetes mellitus. To further explore interactions between CRP and glucose handling, we evaluated the effects of CRP infusion on glucose metabolism in humans. Seven healthy white male volunteers (age, 39.3 +/- 16.9 years) received a single bolus infusion of 1.25 mg/kg purified recombinant human (rh) CRP or CRP-free diluent in a crossover design. C-reactive protein infusion induced an inflammatory response, which was followed by increased plasma concentrations of norepinephrine (3 hours) and cortisol (4 hours). Concomitantly, plasma concentrations of insulin and C-peptide decreased transiently. These metabolic changes increased plasma glucose concentrations from 8 hours after CRP infusion, which was preceded by an increased rate of glucose appearance that was a direct consequence of increased gluconeogenesis. In conclusion, CRP infusion induces an inflammatory response followed by increased norepinephrine and cortisol levels, which results in increased gluconeogenesis. This finding implies that elevated levels of CRP in humans may in fact contribute to altered glucose metabolism and thereby may contribute to the induction of type 2 diabetes mellitus.

High-density lipoprotein attenuates inflammation and coagulation response on endotoxin challenge in humans.

OBJECTIVE: Low high-density lipoprotein (HDL) cholesterol is a strong independent cardiovascular risk factor, which has been attributed to its role in reverse cholesterol transport. Whereas HDL also has potent antiinflammatory effects, the relevance of this property remains to be established in humans. In the present study, we evaluated whether there is a relation between HDL and sensitivity toward a low-dose endotoxin challenge. METHODS AND RESULTS: Thirteen healthy men with genetically determined isolated low HDL cholesterol (averaging 0.7+/-0.1 mmol/L) and 14 age- and body weight-matched healthy men with normal/high HDL cholesterol levels (1.9+/-0.4 mmol/L) were challenged with low-dose endotoxin intravenously (1 ng/kg body weight). The incidence and severity of endotoxin-associated clinical symptoms was increased in the low HDL group. Accordingly, both the inflammatory response (tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, and monocyte chemoattractant protein-1) as well as thrombin generation (prothrombin activation fragments F(1+2)) were significantly increased in the low HDL group on endotoxin challenge. CONCLUSIONS: Low HDL in healthy males is associated with increased sensitivity toward inflammatory stimuli as reflected by enhanced inflammatory and coagulation responses on endotoxin challenge. These antiinflammatory effects of HDL in humans may lend further support to HDL-increasing interventions, particularly in proinflammatory conditions, such as acute coronary syndromes.

Efficacy and safety of high-density lipoprotein cholesterol-increasing compounds: a meta-analysis of randomized controlled trials.

OBJECTIVES: The aim of this research was to estimate the efficacy and safety of current high-density lipoprotein cholesterol (HDL-C)-increasing drugs. BACKGROUND: Epidemiologic evidence has shown that HDL-C is inversely related to coronary heart disease (CHD) risk. However, the evidence for reducing CHD risk by raising HDL-C is thin, predominantly due to the paucity of effective and safe HDL-increasing drugs. METHODS: Randomized controlled trials with fibrates and niacin, published between 1966 through February 2004 (MEDLINE), were retrieved. Information on treatment, baseline characteristics, serum lipids, end points, and side-effects were independently abstracted by two authors using a standardized protocol. RESULTS: Data from 53 trials (16,802 subjects) using fibrates and 30 trials (4,749 subjects) using niacin were included. Random-effects model showed 11% versus 10% reduction in total cholesterol, 36% versus 20% reduction in triglycerides, 8% versus 14% reduction in low-density lipoprotein cholesterol, and 10% versus 16% increase in HDL-C for fibrates and niacin, respectively. Apart from flushes in the niacin group, both fibrates and niacin were shown to be well-tolerated and safe. Fibrates reduced the risk for major coronary events by 25% (95% confidence interval 10% to 38%), whereas current available data for niacin indicate a 27% reduction. CONCLUSIONS: Fibrates reduce major coronary events and increase HDL-C levels without significant toxicity. Niacin has a more potent effect on HDL-C levels, whereas data on cardiovascular event rate reduction are limited. Future studies need to evaluate whether additional HDL increase by fibrates or particularly newer niacin formulations on top of statin therapy translates into further event reduction in high-risk subjects, without significant toxicity.

Loss of heterozygosity and immunohistochemistry of adenocarcinomas of the esophagus and gastric cardia.

PURPOSE: Adenocarcinomas of the distal esophagus and gastric cardia are two tumors that have many features in common. They have similar prognoses, treatment modalities, and patterns of dissemination. The etiology is different, with gastroesophageal reflux disease playing a major role for esophageal adenocarcinoma, in contrast to adenocarcinoma of the gastric cardia. In the present study, we investigated several genetic and immunohistochemical features of adenocarcinomas of the distal esophagus and gastric cardia. EXPERIMENTAL DESIGN: Sixty-two resection specimens of either adenocarcinoma of the esophagus or adenocarcinoma of the gastric cardia were carefully selected. The genetic analysis included loss of heterozygosity of several tumor suppressor genes known to be involved in esophagogastric carcinogenesis. Immunohistochemical studies included the analysis of p53, c-Met, c-erbB-2, beta-catenin, and cyclooxygenase-2. In addition, a mutation analysis of the Tcf1 gene was done by direct sequencing. RESULTS: Patients with cardiac carcinoma had a significantly worse tumor stage and poorer differentiation on histology. Loss of heterozygosity analysis did not reveal significant differences between esophageal adenocarcinoma and cardiac adenocarcinoma. Immunohistochemical analysis revealed significantly more nuclear accumulation of beta-catenin and overexpression of cyclooxygenase-2 in patients with esophageal adenocarcinoma, compared with patients with cardiac carcinoma. No mutation was found in the Tcf1 gene in either tumor type. CONCLUSIONS: Although adenocarcinomas of the distal esophagus and gastric cardia have many features in common, we have found some evidence that they might form two different entities.