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(affects Type 1 AND Type 2 Diabetics)

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Pathophysiology

Interleukin-1–Receptor Antagonist in Type 2 Diabetes Mellitus (NEJM 356:1517-1526, 2007)Background The expression of interleukin-1–receptor antagonist is reduced in pancreatic islets of patients with type 2 diabetes mellitus, and high glucose concentrations induce the production of interleukin-1 in human pancreatic beta cells, leading to impaired insulin secretion, decreased cell proliferation, and apoptosis. Methods In this double-blind, parallel-group trial involving 70 patients with type 2 diabetes, we randomly assigned 34 patients to receive 100 mg of anakinra (a recombinant human interleukin-1–receptor antagonist) subcutaneously once daily for 13 weeks and 36 patients to receive placebo. At baseline and at 13 weeks, all patients underwent an oral glucose-tolerance test, followed by an intravenous bolus of 0.3 g of glucose per kilogram of body weight, 0.5 mg of glucagon, and 5 g of arginine. In addition, 35 patients underwent a hyperinsulinemic–euglycemic clamp study. The primary end point was a change in the level of glycated hemoglobin, and secondary end points were changes in beta-cell function, insulin sensitivity, and inflammatory markers. Results At 13 weeks, in the anakinra group, the glycated hemoglobin level was 0.46 percentage point lower than in the placebo group (P=0.03); C-peptide secretion was enhanced (P=0.05), and there were reductions in the ratio of proinsulin to insulin (P=0.005) and in levels of interleukin-6 (P<0.001) and C-reactive protein (P=0.002). Insulin resistance, insulin-regulated gene expression in skeletal muscle, serum adipokine levels, and the body-mass index were similar in the two study groups. Symptomatic hypoglycemia was not observed, and there were no apparent drug-related serious adverse events. Conclusions The blockade of interleukin-1 with anakinra improved glycemia and beta-cell secretory function and reduced markers of systemic inflammation. (ClinicalTrials.gov number, NCT00303394 [ClinicalTrials.gov] )

• The Inflammation Link: Association between psoriasis, diabetes mellitus, and atherosclerosis - A case-control study(JAAD 56: 629-634 ,April 2007) Background Previous reports demonstrated an association between psoriasis and other diseases including heart failure and diabetes mellitus.Objectives Our aim was to describe the association between psoriasis, diabetes mellitus, and atherosclerosis in Israel. Methods A cross-sectional study was performed utilizing the database of Maccabi Healthcare Services (MHS), a large health provider organization in Israel. Case patients were defined as subjects who were diagnosed with psoriasis. Patients with diabetes and atherosclerosis were identified by using the MHS diabetes and cardiovascular registries, respectively. The control group included MHS enrollees without psoriasis. The proportion of diabetes and atherosclerosis among case and control groups was compared. Chi-square tests were used to compare categorical parameters. Logistic regression models were used for multivariate analyses. Results The study included 46,095 patients with psoriasis (case patients) and 1,579,037 subjects without psoriasis (control patients). The age-adjusted proportion of diabetes was significantly higher in psoriasis patients as compared with the control group (odds ratio [OR] 1.27, 95% confidence interval [CI] 1.1-1.48). The age-adjusted proportion of atherosclerosis was significantly higher in psoriasis patients as compared with the control group (OR 1.28, 95% CI 1.04-1.59). In patients with psoriasis, a multivariate logistic regression model demonstrated an association between diabetes and the multiple use of very potent topical steroids (P < .05) or use of systemic medication for psoriasis (methotrexate, cyclosporine or acitretin) (P < .001). A similar model demonstrated an association between atherosclerosis and the use of phototherapy (P < .001). Limitations Our study was based on a computerized database. The diagnosis of psoriasis was based on digitally transmitted data. Therefore overestimation (false-positive cases) and underestimation (false-negative cases) of psoriasis patients may exist, thereby being a source for information bias. A second limitation is selection bias that may occur due to the possibility that reporting of both psoriasis and associated illnesses is higher in individuals who are seeking medical care. A third limitation concerns the causal effect between occurrence of psoriasis and atherosclerosis or diabetes. The dataset of MHS records diagnoses only from 1997 and does not record the date of disease onset. Conclusions Our study supports previous reports for an association between psoriasis and atherosclerosis and psoriasis and diabetes. Further study is needed to support this observation.

Insulin resistance measured by the euglycaemic insulin clamp predicts subsequent CHD in elderly men. Proinsulin provides a better prediction of CHD than insulin. (Diabetologia [2005]48:1432-0428 (Online))  Aims/hypothesis  The association between CHD and insulin sensitivity (Si) measured by the euglycaemic insulin clamp has not been examined previously. Earlier studies found a relationship between CHD and elevated plasma insulin, an analysis that may have been confounded by co-determination of proinsulin, which has evolved as a stronger predictor of CHD. The aim was to determine the longitudinal relationships between Si, intact proinsulin, 32–33 split proinsulin, specific insulin and subsequent CHD. Methods  This was a population-based cohort study of 815 men in Uppsala, Sweden, aged 70 years at baseline with a follow-up of up to 10 years. Baseline insulin sensitivity was determined by euglycaemic insulin clamp. Fasting proinsulin, 32–33 split proinsulin and specific insulin concentrations were analysed using specific two-site immunometric assays. CHD was taken as diagnosed, if stated (in the event of death) on the Cause of Death Registry, or for subjects hospitalised for the first time with CHD, if CHD was recorded in the Hospital-Discharge Registry. The associations were analysed using Cox's proportional hazards, presented as hazard ratios (HRs) with their 95% CIs for a one-SD increase in the predictor. Results  In multivariate analysis, Si (HR:0.80, CI:0.65–0.97) adjusted for serum cholesterol, systolic blood pressure, fasting plasma glucose, BMI and smoking predicted CHD. Intact proinsulin (HR:1.18, CI:1.01–1.38), adjusted as the model above, predicted CHD, whereas 32–33 split proinsulin (HR:1.13, CI:0.95–1.35) or specific insulin (HR:1.07, CI:0.89–1.30) did not. Conclusions/interpretation  Insulin resistance measured by the euglycaemic insulin clamp predicts subsequent CHD in elderly men. Proinsulin provides a better prediction of CHD than insulin.

• Resistin Is an Inflammatory Marker of Atherosclerosis in Humans (Circulation [2005]111:932-939.) Background— Resistin, a plasma protein, induces insulininsulin-resistant rodents and resistance in rodents. Recent reports suggest that circulating levels of resistin are elevated in obese and humans. Whereas rodent resistin is made in adipocytes,adipocyte and macrophage macrophages are a major source of human resistin. Given the convergence of function, resistin mayResults— We examined provide unique insight into links between obesity, inflammation, and atherosclerosis in humans. Methods and whether plasma resistin(CAC), a quantitative index levels were associated with metabolic and inflammatory markers, as well as with coronary artery calcification of atherosclerosis, in 879 asymptomatic subjects in the Study of Inherited Risk of Coronary Atherosclerosis. Resistin levels were positively associated with levels of inflammatoryand lipoprotein-associated phospholipase markers, including soluble tumor necrosis factor-alpha receptor-2 (P<0.001), interleukin-6 (P=0.04), Aratio and 95% confidence interval in ₂ (P=0.002), but not measures of insulin resistance in multivariable analysis. Resistin levels also were associated (odds ordinal regression)1.52], P=0.03) and further control for with increasing CAC after adjustment for age, sex, and established risk factors (OR, 1.23 [CI, 1.03 to metabolic syndrome and plasma C-reactive proteinmetabolic syndrome, resistin levels (CRP) levels (OR, 1.25 [CI, 1.04 to 1.50], P=0.01). In subjects with further predicted CAC,inflammation and are predictive of whereas CRP levels did not. Conclusions— Plasma resistin levels are correlated with markers of coronary atherosclerosisinflammation, and atherosclerosis. in humans, independent of CRP. Resistin may represent a novel link between metabolic signals, Further studies are needed to define the relationship of resistin to clinical cardiovascular disease.

Tumor Necrosis Factor-alpha Receptor 1 Is a Major Predictor of Mortality and New-Onset Heart Failure in Patients With Acute Myocardial Infarction(Circulation [2005]111:863-870)Background— Tumor necrosis factor alpha (TNF-alpha) activation is an independent prognostic indicator of mortality in patients with heart failure (HF). Despite the recognition that several TNF family cytokines are elevated during myocardial infarction, their role in predicting subsequent prognosis in these setting remains poorly understood. Methods and Results— We performed a systematic evaluation of TNF-alpha and its type 1 and 2 soluble receptors, together with interleukin (IL)-6, IL-1 receptor antagonist, and IL-10, in 184 patients (132 men; mean age, 64±12) consecutively admitted for myocardial infarction. We correlated their values to short- and long-term incidence of death and HF (primary outcome). In 10 patients, we also studied the presence of transcardiac gradients for TNF-alpha and its soluble receptors. The control group comprised 45 healthy subjects who were sex and age matched (33 men; mean age, 65±6 years) to the patients. All tested cytokines were increased in patients, and no transcardiac or systemic AV difference was found. After a median follow-up of 406 days (range, 346 to 696 days), 24 patients died and 32 developed HF. Univariate analysis showed that all cytokines were related to outcome, whereas after adjustment for baseline and clinical characteristics, sTNFR-1 remained the only independent predictor of death and HF (hazard ratio, 2.9; 95% CI, 1.9 to 3.8, tertile 1 versus 3), together with left ventricular ejection fraction, Killip class, and creatine kinase-MB at peak. Conclusions— sTNFR-1 is a major short- and long-term predictor of mortality and HF in patients with acute myocardial infarction.

• Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species (Am J Physiol Heart Circ Physiol [2005]288:H854-H860)Insulin resistance (IR) and associated hyperinsulinemia are major risk factors for coronary artery disease. Mechanisms linking hyperinsulinemia to coronary vascular dysfunction in IR are unclear. We evaluated insulin-induced vasodilation in isolated small coronary arteries (SCA; ~225 µm) of Zucker obese (ZO) and control Zucker lean (ZL) rats. Vascular responses to insulin (0.1–100 ng/ml), ACh (10^–9–10^–5 mol/l), and sodium nitroprusside (10^–8–10^–4 mol/l) were assessed in SCA by measurement of intraluminal diameter using videomicroscopy. Insulin-induced dilation was decreased in ZO compared with ZL rats, whereas ACh and sodium nitroprusside elicited similar vasodilations. Pretreatment of arteries with SOD (200 U/ml), a scavenger of reactive oxygen species (ROS), restored the vasorelaxation response to insulin in ZO arteries, whereas ZL arteries were unaffected. Pretreatment of SCA with N-nitro-L-arginine methyl ester (100 µmol/l), an inhibitor of endothelial nitric oxide (NO) synthase (eNOS), elicited a vasoconstrictor response to insulin that was greater in ZO than in ZL rats. This vasoconstrictor response was reversed to vasodilation in ZO and ZL rats by cotreatment of the SCA with SOD or apocynin (10 µmol/l), a specific inhibitor of vascular NADPH oxidase. Lucigenin-enhanced chemiluminescence showed increased basal ROS levels as well as insulin (330 ng/ml)-stimulated production of ROS in ZO arteries that was sensitive to inhibition by apocynin. Western blot analysis revealed increased eNOS expression in ZO rats, whereas Mn SOD and Cu,Zn SOD expression were similar to ZL rats. Thus IR in ZO rats leads to decreased insulin-induced vasodilation, probably as a result of increased production of ROS by vascular NADPH oxidase, leading to decreased NO bioavailability, despite a compensatory increase in eNOS expression.

Circulating Mononuclear Cells In The Obese Found To Be In Proinflammatory State, Contributing To Diabetes And Heart Disease (Circulation [2004] 110:1564-1571) Background— In view of the increase in plasma concentrations of proinflammatory mediators tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and C-reactive protein (CRP) in obesity, we investigated whether peripheral blood mononuclear cells (MNC) from obese subjects are in a proinflammatory state. Methods and Results— MNC were prepared from fasting blood samples of obese (n=16; body mass index [BMI]=37.7±5.0 kg/m²) and normal-weight control (n=16; BMI=23.8±1.9 kg/m²) subjects. Nuclear factor kB (NF-kB) binding to DNA in nuclear extracts was elevated (P<0.05) and the inhibitor of NFkB-ß (IkB-ß) was significantly lower (P<0.001) in the obese group. Reverse transcription–polymerase chain reaction revealed elevated levels of migration inhibitor factor (MIF), IL-6, TNF-a, and matrix metalloproteinase-9 (MMP-9) mRNA expression in the obese subjects (P<0.05). Plasma concentrations of MIF, IL-6, TNF-a, MMP-9, and CRP were also significantly higher. Plasma glucose, insulin, and free fatty acids (FFAs) were measured, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Plasma FFA concentration related significantly to BMI, IL-6, and TNF-a mRNA expression and plasma CRP levels but not to HOMA-IR. On the other hand, the inflammatory mediators were significantly related to BMI and HOMA-IR. Conclusions— These data show (1) for the first time that MNC in obesity are in a proinflammatory state with an increase in intranuclear NF-kB binding, a decrease in IkB-ß, and an increase in the transcription of proinflammatory genes regulated by NF-kB; (2) that plasma FFAs are a modulator of inflammation; and (3) that insulin resistance is a function of inflammatory mediators.

• Urinary 20-Hydroxyeicosatetraenoic Acid Is Associated With Endothelial Dysfunction in Humans (Circulation [2004]110:438-443) Background— 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 (-hydroxylase) metabolite of arachidonic acid with vasoconstrictor activity that may be involved in the pathogenesis of hypertension. In humans, there are few data relating 20-HETE to vascular pathophysiology. This study aimed to determine whether urinary 20-HETE excretion is related to blood pressure or vascular endothelial function in humans. Methods and Results— Sixty-six subjects (37 males, 29 females), including 29 with untreated hypertension, had urinary 20-HETE excretion measured by gas chromatography/mass spectrometry. There was no significant difference for 20-HETE excretion between hypertensive and normotensive subjects. 20-HETE excretion was positively related to body mass index and sodium excretion. There was a significant inverse association between urinary 20-HETE and endothelium-dependent vasodilation measured by flow-mediated dilation of the brachial artery (P=0.006). There was no association with vasodilator responses to nitroglycerin. In multiple regression analysis, 20-HETE remained an independent predictor of endothelium-dependent vasodilation after adjustment for age, body mass index, and blood pressure. When gender was included in the model, the relationship between 20-HETE and flow-mediated dilation was attenuated. Separate analysis by gender revealed that in women, hypertensive subjects had significantly higher 20-HETE excretion than normotensive subjects, but this was not seen in men. In women, 20-HETE was positively related to diastolic and systolic blood pressure. In men, 20-HETE was positively related to body mass index. Conclusions— This is the first demonstration of an association between 20-HETE excretion and in vivo vascular function in humans. Given the negative modulatory role of nitric oxide on -hydroxylase, the present results suggest a potentially important role for 20-HETE in human vascular physiology.

Fish intake may influence risk of Atrial Fibrillation (Circulation [2004]110:368-373) Background— Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is particularly common in the elderly. Although effects of fish intake, including potential antiarrhythmic effects, may favorably influence risk of AF, relationships between fish intake and AF incidence have not been evaluated. Methods and Results— In a prospective, population-based cohort of 4815 adults >=age 65 years, usual dietary intake was assessed at baseline in 1989 and 1990. Consumption of tuna and other broiled or baked fish correlated with plasma phospholipid long-chain n-3 fatty acids, whereas consumption of fried fish or fish sandwiches (fish burgers) did not. AF incidence was prospectively ascertained on the basis of hospital discharge records and annual electrocardiograms. During 12 years’ follow-up, 980 cases of incident AF were diagnosed. In multivariate analyses, consumption of tuna or other broiled or baked fish was inversely associated with incidence of AF, with 28% lower risk with intake 1 to 4 times per week (HR=0.72, 95% CI=0.58 to 0.91, P=0.005), and 31% lower risk with intake >=5 times per week (HR=0.69, 95% CI=0.52 to 0.91, P=0.008), compared with <1 time per month (P trend=0.004). Results were not materially different after adjustment for preceding myocardial infarction or congestive heart failure. In similar analyses, fried fish/fish sandwich consumption was not associated with lower risk of AF.Conclusions— Among elderly adults, consumption of tuna or other broiled or baked fish, but not fried fish or fish sandwiches, is associated with lower incidence of AF. Fish intake may influence risk of this common cardiac arrhythmia.

• Data suggest that AKT may be involved in the endothelial dysfunction of hypertension (Circulation [2004]109:2587-2593) Background— In hypertension, reduced nitric oxide production and blunted endothelial vasorelaxation are observed. It was recently reported that AKT phosphorylates and activates endothelial nitric oxide synthase and that impaired kinase activity may be involved in endothelial dysfunction. Methods and Results— To identify the physiological role of the kinase in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), we used adenoviral vectors to transfer the human AKT1 gene selectively to the common carotid endothelium. In vitro, endothelial vasorelaxations to acetylcholine, isoproterenol, and insulin were blunted in control carotids from SHR compared with WKY rats, and human AKT1 overexpression corrected these responses. Similarly, blood flow assessed in vivo by Doppler ultrasound was reduced in SHR compared with WKY carotids and normalized after AKT1 gene transfer. In primary cultured endothelial cells, we evaluated AKT phosphorylation, activity, and compartmentalization and observed a mislocalization of the kinase in SHR. Conclusions— We conclude that AKT participates in the settings of endothelial dysfunction in SHR rats by impaired membrane localization. Our data suggest that AKT is involved in endothelium dysfunction in hypertension.

Oxidative stress induces insulin resistance by activating the nuclear factor-kB pathway and disrupting normal subcellular distribution of phosphatidylinositol 3-kinase (Diabetologia [2004] 47: 794 - 805) Aims/hypothesis  Oxidative stress is associated with diabetes, hypertension and atherosclerosis. Insulin resistance is implicated in the development of these disorders. We tested the hypothesis that oxidative stress induces insulin resistance in rats, and endeavoured to identify mechanisms linking the two. Methods  Buthionine sulfoximine (BSO), an inhibitor of glutathione synthase, was administered to Sprague-Dawley rats and 3T3-L1 adipocytes. Glucose metabolism and insulin signalling both in vivo and in 3T3-L1 adipocytes were examined. In 3T3-L1 adipocytes, the effects of overexpression of a dominant negative mutant of inhibitory kB (IkB), one role of which is to block oxidative-stress-induced nuclear factor (NF)-kB activation, were investigated. Results  In rats given BSO for 2 weeks, the plasma lipid hydroperoxide level doubled, indicating increased oxidative stress. A hyperinsulinaemic-euglycaemic clamp study and a glucose transport assay using isolated muscle and adipocytes revealed insulin resistance in BSO-treated rats. BSO treatment also impaired insulin-induced glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. In BSO-treated rat muscle, adipose tissue and 3T3-L1 adipocytes, insulin-induced IRS-1 phosphorylation in the low-density microsome (LDM) fraction was specifically decreased, while that in whole cell lysates was not altered, and subsequent translocation of phosphatidylinositol (PI) 3-kinase from the cytosol and the LDM fraction was disrupted. BSO-induced impairments of insulin action and insulin signalling were reversed by overexpressing the dominant negative mutant of IkB, thereby suppressing NF-kB activation. Conclusions/interpretation  Oxidative stress induces insulin resistance by impairing IRS-1 phosphorylation and PI 3-kinase activation in the LDM fraction, and NF-kB activation is likely to be involved in this process.

• Endothelial dysfunction predicts type 2 diabetes in women independent of other known risk factors, including obesity and subclinical inflammation.(JAMA [2004] 291:1978-1986) Context  Endothelial dysfunction occurs in diagnosed type 2 diabetes mellitus but may also precede development of diabetes. Objective  To determine whether elevated plasma levels of biomarkers reflecting endothelial dysfunction (E-selectin; intercellular adhesion molecule 1 [ICAM-1]; and vascular cell adhesion molecule 1 [VCAM-1]) predict development of type 2 diabetes in initially nondiabetic women. Design and Setting  Prospective, nested case-control study within the Nurses' Health Study, an ongoing US study initiated in 1976. Participants  Of 121 700 women initially enrolled, 32 826 provided blood samples in 1989-1990; of those free of diabetes, cardiovascular disease, or cancer at baseline, 737 developed incident diabetes by 2000. Controls (n = 785) were selected according to matched age, fasting status, and race. Main Outcome Measure  Risk of confirmed clinically diagnosed type 2 diabetes by baseline levels of E-selectin, ICAM-1, and VCAM-1. Results  Baseline median levels of the biomarkers were significantly higher among cases than among controls (E-selectin, 61.2 vs 45.4 ng/mL; ICAM-1, 264.9 vs 247.0 ng/mL; VCAM-1, 545.4 vs 526.0 ng/mL [all P values <=0.004]). Elevated E-selectin and ICAM-1 levels predicted incident diabetes in logistic regression models conditioned on matching criteria and adjusted for body mass index (BMI), family history of diabetes, smoking, diet score, alcohol intake, activity index, and postmenopausal hormone use. The adjusted relative risks for incident diabetes in the top quintile vs the bottom quintiles were 5.43 for E-selectin (95% confidence interval [CI], 3.47-8.50), 3.56 for ICAM-1 (95% CI, 2.28-5.58), and 1.12 for VCAM-1 (95% CI, 0.76-1.66). Adjustment for waist circumference instead of BMI or further adjustment for baseline levels of C-reactive protein, fasting insulin, and hemoglobin A_1c or exclusion of cases diagnosed during the first 4 years of follow-up did not alter these associations. Conclusion  Endothelial dysfunction predicts type 2 diabetes in women independent of other known risk factors, including obesity and subclinical inflammation.

Interleukin-6 is a positive regulator of tumor necrosis factor -induced adipose-related protein in 3T3-L1 adipocytes (FEBS Letters [2004] 560:153-157) Tumor necrosis factor (TNF)alpha induced adipose-related protein (TIARP) is a novel TNF-alpha-stimulated protein in adipocytes. Besides TNFalpha, interleukin (IL)-6 has recently been shown to be another adipocytokine implicated in insulin resistance. Therefore, the impact of IL-6 on TIARP gene expression in 3T3-L1 adipocytes was determined by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, TIARP mRNA expression was stimulated up to 3.8-fold by IL-6 in a dose-dependent fashion with significant stimulation detectable at effector concentrations as low as 3 ng/ml and maximal effects seen at 100 ng/ml IL-6. Induction of TIARP mRNA by IL-6 was time-dependent with significant upregulation occurring as early as 2 h after effector addition and maximal effects observed at 4 h. In parallel, TIARP protein synthesis was upregulated with maximal effects seen after 8 h of IL-6 treatment. Furthermore, the Janus kinase 2 inhibitor AG490 decreased TIARP mRNA expression. The increase of TIARP mRNA could be reversed by withdrawal of IL-6 for 24 h. Furthermore, TIARP mRNA induction by IL-6 was also seen in brown adipocytes but not in muscle and liver cells. Taken together, these results show that TIARP is acutely regulated in adipose tissue not only by TNFalphabut also by IL-6 which has been shown to be another important cytokine implicated in the pathogenesis of insulin resistance.

• Plasminogen activator inhibitor correlates with coronary wall thickening in patients with angiographically normal coronary arteries (Thrombosis Research [2003] 112: 123-129) Introduction: Angiographically normal coronary arteries have concealed intimal thickening that importantly contribute to coronary arterial disease activity. Increased plasma levels of plasminogen activator inhibitor (PAI) are associated with myocardial infarction and atherosclerosis. However, it remains unclear whether the PAI contributes to vascular wall thickening detected by intravascular ultrasound (IVUS) in normal coronary angiogram. The aim of this study was to evaluate if the PAI activity contributes to the extent of atherosclerotic changes in angiographically normal coronary arteries using IVUS technique. Materials and methods: We studied 33 consecutive patients with normal coronary angiograms. These patients were divided into a high level of plasma PAI activity group (H-PAI; n=12) and a normal range of PAI activity group (N-PAI; n=21), according to the plasma PAI activity levels. Results: The average of "percent intima+media area (%I+M area)" and "maximal intima+media (I+M) thickness" were significantly greater in the H-PAI group as compared with those in the N-PAI group (p<0.05). Minimal lumen diameter and lumen area were comparable between these groups. The plasma PAI activity level was the independent predictor of increase in maximal I+M thickness, in multiple regression analysis with the traditional risk factors as covariates. Conclusions: Thickened intima+media of angiographically normal coronary arteries were associated with high plasma level of PAI activity, independently of other traditional risk factors. PAI may contribute to the pathogenesis of coronary intimal thickening that might increase coronary arterial tone. Author Keywords: Plasminogen activator inhibitor; Coronary wall thickening; Intravascular ultrasound; Normal coronary angiogram; Quantitative coronary angiography; Nitric oxides

Beta-blockers enhance natriuretic peptides' effects during exercise (J Am Coll Cardiol [2004] 43:353-359) In patients with coronary artery disease, natriuretic peptide levels in plasma are increased during exercise, French physicians now show that treatment with beta-blockers enhances the natriuretic peptide release, thus decreasing cardiac load.Cardiac disease is associated with increased resting levels of atrial and brain natriuretic peptide. Under stress, cardiomyocytes release both atrial and brain natriuretic peptide, which exert vasodilator and diuretic effects, which many be expected to reduce cardiac load. However, this process in patients with coronary artery disease has been poorly characterized.Therefore, Dr. Pierre-Yves Marie and colleagues at UPRES EA in Nancy, measured natriuretic peptide levels at rest and peak exercise in 104 patients with chronic coronary artery disease. They describe their findings in the Journal of the American College of Cardiology for February 4th.The best independent predictors of natriuretic peptide concentrations at rest were aging and the extent of scarred left ventricular area. "No previous study has given evidence of the dramatic influence of the extent of scarred myocardium, as determined by myocardial SPECT," the authors write.Beta blocker treatment did not affect natriuretic peptide concentrations at rest. However, during exercise, the average increase in both atrial and brain natriuretic peptide was more than doubled in the 55 patients treated with beta-blockers, even though their heart rate did not increase as much."This enhanced secretion of potent vasodilating and natriuretic agents might constitute an additional and original mechanism of these drugs for further protecting diseased hearts against stress," Dr. Marie's group concludes.

• MCC134 - which inhibits preconditioning - opens cardiac surface k(ATP) channels but blocks mito k(ATP) channels reaffirming the dependency of cardioprotection upon mito rather than surface k(ATP) channels (Circulation [2003]107(8):1183-8)BACKGROUND: MCC-134 (1-[4-(H-imidazol-1-yl)benzoyl] -N-methylcyclobutane-carbothioamide), a newly developed analog of aprikalim, opens surface smooth muscle-type ATP-sensitive potassium (K(ATP)) channels but inhibits pancreatic K(ATP) channels. However, the effects of MCC-134 on cardiac surface K(ATP) channels and mitochondrial K(ATP) (mitoK(ATP)) channels are unknown. A mixed agonist/blocker with differential effects on the two channel types would help to clarify the role of K(ATP) channels in cardioprotection. METHODS AND RESULTS: To index mitoK(ATP) channels, we measured mitochondrial flavoprotein fluorescence in rabbit ventricular myocytes. MCC-134 alone had little effect on basal flavoprotein fluorescence. However, MCC-134 inhibited diazoxide-induced flavoprotein oxidation in a dose-dependent manner (EC(50)=27 micro mol/L). When ATP was included in the pipette solution, MCC-134 slowly activated surface K(ATP) currents with some delay (>10 minutes). These results indicate that MCC-134 is a mitoK(ATP) channel inhibitor and a surface K(ATP) channel opener in native cardiac cells. In cell-pelleting ischemia assays, coapplication of MCC-134 with diazoxide abolished the cardioprotective effect of diazoxide, whereas MCC-134 alone did not alter cell death. These results were reproducible in both rabbit and mouse myocytes. MCC-134 also attenuated the effect of ischemic preconditioning against myocardial infarction in mice, consistent with the results of cell-pelleting ischemia assays. CONCLUSIONS: A single drug, MCC-134, opens surface K(ATP) channels but blocks mitoK(ATP) channels; the fact that this drug inhibits preconditioning reaffirms the primacy of mitoK(ATP) rather than surface K(ATP), channels in the mechanism of cardioprotection.

Stress upregulates arterial matrix metalloproteinase expression and activity via endothelin 1-A receptor activation (Am J Physiol Heart Circ Physiol [2003] 285: H2225 - H2232) Degradation of the extracellular matrix proteins by matrix metalloproteinases (MMP) is an important regulatory step in the vascular remodeling process. Recent studies demonstrated that ET_A receptors regulate cardiac MMP activity and fibrosis in DOCA-salt hypertension. However, little is known about endothelin (ET)-1 regulation of vascular MMP activity in hypertension. Thus early changes in ET-1-mediated regulation of MMP activity were measured in borderline hypertensive rats that develop impaired vasorelaxation and hypertension with chronic exposure to stress. Experiments were performed after 10 days of exposure to the behavioral stressor, air-jet stress, but before the onset of stress-induced hypertension. Study groups were 1) control (n = 8); 2) air-jet stress for 10 days (n = 8); 3) control plus ET_A antagonist ABT-627 (n = 4), and 4) air-jet stress plus ET_A antagonist (n = 4). MMP activity in the thoracic aorta was assessed by gelatin zymography. MMP protein and tissue ET-1 levels were evaluated by immunohistochemistry, and ET receptor density was determined by immunoblotting. Exposure to stress caused a twofold increase in plasma ET-1 levels (P < 0.05), and there was increased ET-1 staining at the tissue level. Total MMP activity and expression of MMP-2 and MMP-9 were increased in the stress group. ET_A receptor antagonism prevented the increase in MMP expression and activation in the stress group. These results provide evidence that the MMP system is activated before the development of hypertension and ET-1 mediates these early events in vascular remodeling.

• Inhibition of NF-kB activation in macrophages increases atherosclerosis in LDL receptor–deficient mice (J. Clin. Invest.[2003]112:1176-1185 doi:10.1172/JCI200318580) Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kB activation in macrophages during atherogenesis, we used LDL receptor–deficient mice with a macrophage-restricted deletion of IkB kinase 2 (IKK2), which is essential for NF-kB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Tumor Necrosis Factor-alpha Inhibits Insulin’s Stimulating Effect on Glucose Uptake and Endothelium-Dependent Vasodilation in Humans (Circulation [2003]108:1815) Background— Inflammatory mechanisms could be involved in the pathogenesis of both insulin resistance and atherosclerosis. Therefore, we aimed at examining whether the proinflammatory cytokine tumor necrosis factor (TNF)-alpha inhibits insulin-stimulated glucose uptake and insulin-stimulated endothelial function in humans. Methods and Results— Healthy, lean male volunteers were studied. On each study day, 3 acetylcholine (ACh) or sodium nitroprusside (SNP) dose-response studies were performed by infusion into the brachial artery. Before and during the last 2 dose-response studies, insulin and/or TNF-alpha were coinfused. During infusion of insulin alone for 20 minutes, forearm glucose uptake increased by 220±44%. This increase was completely inhibited during coinfusion of TNF-alpha (started 10 min before insulin) with a more pronounced inhibition of glucose extraction than of blood flow. Furthermore, TNF-alpha inhibited the ACh forearm blood flow response (P<0.001), and this inhibition was larger during insulin infusion (P=0.01) but not further increased by N^G-monomethyl-L-arginine acetate (P=0.2). Insulin potentiated the SNP response less than the ACh response and the effect of TNF-alpha was smaller (P<0.001); TNF-alpha had no effect on the SNP response without insulin infusion. Thus, TNF-alpha inhibition of the combined response to insulin and ACh was likely mediated through inhibition of NO production. ^f Conclusion— These results support the concept that TNF-^alpha could play a role in the development of insulin resistance in humans, both in muscle and in vascular tissue.

• Atherosclerosis is well-begun preceding the clinical onset of diabetes (Arteriosclerosis, Thrombosis, and Vascular Biology [2003] 23:1845) Objective— We examined whether B-mode ultrasound–detected carotid artery intima-media thickness (IMT) was elevated before the onset of clinical diabetes. Methods and Results— The study population for these analyses included 1127 nondiabetic participants, 66 prediabetic participants, and 303 diabetic participants with a mean age of 49.8 years who participated in the Mexico City Diabetes Study, a prospective cohort study. Common carotid artery (CCA) and internal carotid artery (ICA) IMTs were measured bilaterally by B-mode ultrasound. Age- and sex-adjusted mean ICA and CCA IMTs were both significantly higher among prediabetic individuals {0.81 mm [95% confidence interval (CI), 0.75–0.88] and 0.72 mm [95% CI, 0.69–0.75], respectively} than in individuals who remained free of diabetes [0.71 mm (95% CI, 0.69–0.72) and 0.69 mm (95% CI, 0.68–0.69), respectively]. However, after adjustment for established cardiovascular risk factors, ICA IMT, but not CCA IMT, remained significantly higher among prediabetic individuals [0.81 mm (95% CI, 0.75–0.88) and 0.71 mm (95% CI, 0.68–0.74)] than in individuals who remained free of diabetes [0.71 mm (95% CI, 0.69–0.72) and 0.69 mm (95% CI, 0.68–0.70)]. Conclusions— The present study provides direct evidence at the vascular level that atherosclerosis levels are elevated before the clinical onset of diabetes.

Stearoyl-CoA desaturase 1 deficiency elevates insulin-signaling components and down-regulates protein-tyrosine phosphatase 1B in muscle (PNAS [2003] 100:11110-11115)

• Macrophage-Specific p53 Expression Plays a Crucial Role in Atherosclerosis Development and Plaque Remodeling (Arteriosclerosis, Thrombosis, and Vascular Biology [2003]23:1608) Objective— We first showed that absence of p53 accelerates atherosclerosis development in apoE-deficient mice. In this study, we investigated how macrophage-specific loss of p53 function might modulate atherosclerosis development in LDL receptor-deficient mice, a model for familial hypercholesterolemia. Methods and Results— We transferred bone marrow cells isolated from p53^+/+ and p53^-/- mice to lethally irradiated LDL receptor^-/- mice and evaluated the aortic atherosclerotic lesion areas in the recipients at different times afterward. At 15 weeks and again at 20 weeks, we found larger aortic lesion size in mice receiving p53^-/- cells compared with those that received p53^+/+ cells. By measuring the rate of bromodeoxyuridine incorporation, we found that the absence of p53 in macrophages stimulates cellular proliferation. In contrast, the rate of apoptosis in the atheromatous lesion was similar in the two groups of mice. Furthermore, we found that the absence of macrophage-specific p53 expression was associated with vulnerable-appearing lesions marked by increased tissue necrosis and reduced collagen deposition. Conclusions— p53 plays a crucial role in atherosclerosis lesion development and remodeling, and macrophage-specific p53 deficiency stimulates cellular proliferation leading to a vulnerable-appearing phenotype of lesions in a mouse model of familial hypercholesterolemia. Key Words: atherosclerosis • apoptosis • proliferation • necrosis • macrophage

IGT and DM2 associate with increased arterial stiffness occurring before the onset of DM2 and explained neither by conventional CV risk factors nor by hyperglycemia nor by hyperinsulinemia (Circulation [2003]107:2089.) Background— Type 2 diabetes (DM-2) and impaired glucose metabolism (IGM) are associated with an increased cardiovascular disease risk. In nondiabetic individuals, increased arterial stiffness is an important cause of cardiovascular disease. Associations between DM-2 and IGM and arterial stiffness have not been systematically investigated. Methods and Results— In a population-based cohort (n=747; 278 with normal glucose metabolism, 168 with IGM, and 301 with DM-2; mean age, 68.5 years), arterial stiffness was ultrasonically estimated by distensibility and compliance of the carotid, femoral, and brachial arteries and by the carotid elastic modulus. After adjustment for age, sex, and mean arterial pressure, DM-2 was associated with increased carotid, femoral, and brachial stiffness, whereas IGM was associated only with increased femoral and brachial stiffness. Carotid but not femoral or brachial stiffness increased from IGM to DM-2. Standardized ßs (95% CI) for IGM and DM-2, compared with normal glucose metabolism, were -0.06 (-0.23 to 0.10) and -0.37 (-0.51 to -0.23) for carotid distensibility; -0.02 (-0.18 to 0.18) and -0.25 (-0.40 to -0.09) for carotid compliance; -0.05 (-0.23 to 0.13) and 0.25 (0.10 to 0.40) for carotid elastic modulus; -0.70 (-0.89 to -0.51) and -0.67 (-0.83 to -0.52) for femoral distensibility; and -0.62 (-0.80 to -0.44) and -0.79 (-0.94 to -0.63) for femoral compliance. The brachial artery followed a pattern similar to that of the femoral artery. Increases in stiffness indices were explained by decreases in distension, increases in pulse pressure, an increase in carotid intima-media thickness, and, for the femoral artery, a decrease in diameter. Hyperglycemia or hyperinsulinemia explained only 30% of the arterial changes associated with glucose tolerance. Adjustment for conventional cardiovascular risk factors did not affect these findings. Conclusions— IGM and DM-2 are associated with increased arterial stiffness. An important part of the increased stiffness occurs before the onset of DM-2 and is explained neither by conventional cardiovascular risk factors nor by hyperglycemia or hyperinsulinemia.

• In-hospital LVF and cardiac mortality imply admission glycemia in ACS [independent of diabetic status] (Heart [2003] 24:309-321) Objectives: To analyse the relation between serum glucose concentration and hospital outcome across the whole spectrum of acute coronary syndromes. Methods: This was a prospective cohort study of 2127 patients presenting with acute coronary syndromes. The patients were stratified into quartile groups (Q1 to Q4) defined by serum glucose concentrations of 5.8, 7.2, and 10.0 mmol/l. The relation between quartile group and major in-hospital complications was analysed. Results: The proportion of patients with acute myocardial infarction increased incrementally across the quartile groups, from 21.4% in Q1 to 47.9% in Q4 (p < 0.0001). The trend for frequency of in-hospital major complications was similar, particularly left ventricular failure (LVF) (Q1 6.4%, Q4 25.2%, p < 0.0001) and cardiac death (Q1 0.7%, Q4 6.1%, p < 0.0001). The relations were linear, each glucose quartile increment being associated with an odds ratio of 1.46 (95% confidence interval (CI) 1.27 to 1.70) for LVF and 1.52 (95% CI 1.17 to 1.97) for cardiac death. Although complication rates were higher for a discharge diagnosis of acute myocardial infarction than for unstable angina, there was no evidence that the effects of serum glucose concentration were different for the two groups, there being no significant interaction with discharge diagnosis in the associations between glucose quartile and LVF (p = 0.69) or cardiac death (p = 0.17). Similarly there was no significant interaction with diabetic status in the associations between glucose quartile and LVF (p = 0.08) or cardiac death (p = 0.09). Conclusion: Admission glycaemia stratified patients with acute coronary syndromes according to their risk of in-hospital LVF and cardiac mortality. There was no detectable glycaemic threshold for these adverse effects. The prognostic correlates of admission glycaemia were unaffected by diabetic status and did not differ significantly between patients with acute myocardial infarction and those with unstable angina.

S447X (serine) substitution on lipoprotein lipase (LPL) gene associates frequently with a high-HDL/low-LDL phenotype and infrequently with a low-HDL/high-LDL phenotype (Genetic Epidemiology [2003] 24:309-321) S447X, a serine substitution by a stop codon on base 99 of exon 9 of the lipoprotein lipase (LPL) gene, has beneficial effects on blood lipids. Other LPL alleles are associated with lipid levels, but whether one of these variants predominates remains elusive. We performed a systematic survey to identify single-nucleotide polymorphisms (SNPs) in all 10 LPL exons and flanking regions by resequencing the gene in 95 subjects. Of 24 variants, 14 were common (>=3%). We assayed the common SNPs in 186 cases with atherogenic lipid profiles (low HDL, high LDL) and 185 nonatherogenic controls (high HDL, low LDL). Only S447X and exons 6 (base +73) and 10 (base -11) were individually associated with case-control status (P<0.05, adjusted for major nongenetic covariates with known lipid effects). There were no significant SNP×gender interactions. In adjusted multi-SNP and haplotypic analyses, S447X was interpretable as the sole predictor, with a 2-3-fold reduction in the odds of being atherogenic vs. nonatherogenic (adjusted OR, 0.39; 95% CI, 0.21-0.73). S447X and base -11 of exon 10 were statistically interchangeable because they are strongly associated (r=0.92, P<0.0001), but we posit that the LPL association with lipid profile is more likely attributable to the functional S447X rather than the nonfunctional exon 10 SNP. It appears that the S447X variant of LPL may be another rare example (like APOE4, factor V-Leiden, and PPAR-gamma Pro12Ala) of a common variant predisposing to a common disorder

• Fasting glucose levels appear to increase in dysmetabolic hypertensive patients who develop MI and to decrease in normotensives who do not (BMJ [2003] 326:681) Objective: To investigate the impact of an increase in blood glucose on the risk of developing myocardial infarction, with particular emphasis on people taking antihypertensive drugs. Design: Prospective population based cohort study. Setting: Uppsala, Sweden. Participants: 1860 men who had participated in 1970-3 at age 50 in a health survey aimed at identifying risk factors for cardiovascular disease and were re-examined at age 60 and then followed for 17.4 years. Main outcome measure: Myocardial infarction after age 60. Results: The incidence of myocardial infarction was significantly higher in men treated for hypertension than in those without such treatment (23% v 13.5%, P<0.0001). Participants who developed myocardial infarction after the age of 60 (n=253) showed a significantly larger increase in blood glucose between age 50 and 60 than did those without myocardial infarction. In multivariate Cox proportional hazard models increase in blood glucose was an independent risk factor for myocardial infarction (P=0.0001) in men receiving antihypertensive treatment at age 60 (n=291, mainly beta- blockers and thiazide diuretics) but not in those without such treatment. The impact of increase in blood glucose declined after inclusion of serum proinsulin concentrations at baseline but was still significant. A significant interaction existed between proinsulin concentration (a marker of insulin resistance) at baseline and antihypertensive treatment on increase in blood glucose. Conclusions: Increase in blood glucose between the ages of 50 and 60 and baseline proinsulin concentration were important risk factors for myocardial infarction in men receiving antihypertensive treatment, indicating that both an insulin resistant state and the metabolic impact of beta- blockers and diuretics increase the risk of myocardial infarction.
  

Antileukotriene drugs may be an effective treatment regimen in late-stage atherogenesis (Proc. Natl. Acad. Sci. USA, [2003] 10.1073/pnas.242716099) Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.

• Diabetic Macrovascular Disease -The Glucose Paradox? (Circulation. 2002;106:2760) -editorial by Peter Libby The specific interventions used to lower glycemia may contribute to the inability to show decreases in macrovascular end points. With some antidiabetic treatments, untoward effects may counterbalance potential benefits. Generally, interventions that increase insulin supply (eg, insulin itself and sulfonylureas) have proven less promising for limiting cardiovascular complications than those that improve glucose utilization or reduce insulin resistance. Indeed, in one arm of UKPDS, metformin monotherapy decreased MI by 39% (P0.01) in an overweight subgroup, a benefit not seen in patients requiring metformin plus sulfonylureas or insulin. Thiazolidinediones (the "glitazones") hold considerable promise as insulin sensitizers and merit careful clinical evaluation for cardiovascular benefit.

Elevated levels of acute-phase proteins and plasminogen activator inhibitor-1 predict the development of type 2 diabetes: the insulin resistance atherosclerosis study.(Diabetes 2002 Apr;51(4):1131-7) Festa A, D'Agostino R Jr, Tracy RP, Haffner SM; The Insulin Resistance Atherosclerosis Study.Elevated serum levels of acute-phase proteins, indicating chronic subclinical inflammation, have been associated with cardiovascular disease as well as the insulin resistance syndrome. Chronic inflammation may also be a risk factor for developing type 2 diabetes. We studied the concentrations of C-reactive protein (CRP), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1) in 1,047 nondiabetic subjects in relation to incident diabetes within 5 years in the Insulin Resistance Atherosclerosis Study. Subjects with diabetes at follow-up (n = 144) had higher baseline levels of fibrinogen (mean +/- SD; 287.8 +/- 58.8 vs. 275.1 +/- 56.0 mg/dl; P = 0.013) as well as of CRP (median [interquartile range]; 2.40 [1.29, 5.87] vs. 1.67 mg/l [0.75, 3.41]; P = 0.0001) and PAI-1 (24 [15, 37.5] vs. 16 ng/ml [9, 27]; P = 0.0001) than nonconverters. The odds ratio (OR) of converting to diabetes was significantly increased with increasing baseline concentrations of the inflammatory markers. In contrast to PAI-1, the association of CRP and fibrinogen with incident diabetes was significantly attenuated after adjustment for body fat (BMI or waist circumference) or insulin sensitivity (S(I)), as assessed by a frequently sampled intravenous glucose tolerance test. In a logistic regression model that included age, sex, ethnicity, clinical center, smoking, BMI, S(I), physical activity, and family history of diabetes, PAI-1 still remained significantly related to incident type 2 diabetes (OR [95% CI] for 1 SD increase: 1.61 [1.20-2.16]; P = 0.002). Chronic inflammation emerges as a new risk factor for the development of type 2 diabetes; PAI-1 predicts type 2 diabetes independent of insulin resistance and other known risk factors for diabetes.

• Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase (Arteriosclerosis, Thrombosis, and Vascular Biology 2002, 10.1161/01) Objective: The objective of this study was to investigate whether depolarization of cultured endothelial cells (human umbilical vein endothelial cells, HUVECs) affects their ectonucleotidase activity through superoxide (O2-) production. Methods and Results—Depolarization by the cation channel gramicidin (100 nmol/L) or tetrabutylammonium chloride (1 mmol/L) induced O2- release from HUVECs (n=4), which was decreased by superoxide dismutase (SOD, 500 U/mL). The activity of endothelial ectonucleotidases was assessed by the production of inorganic phosphate from ADP, which was decreased by chronic depolarization by 25% (n=6, P<0.05) and the amount of AMP derived from ADP in the presence of the 5'-nucleotidase inhibitor ,ß-methylene-5'-diphosphate (00 µmol/L). AMP was decreased by chronic depolarization from 0.54±0.16 to 0.39±0.11 µmol/min/mg protein (n=6, P<0.05). This was abolished in the continuous presence of SOD (n=6). NTPDase protein was predominantly expressed in HUVECs (n=4). Protein abundance, viability of cells, and apoptosis rates were not altered by depolarization (n=10). Only in the presence of depolarized HUVECs, but not with control cells or with HUVECs depolarized in the presence of SOD, did 5 µmol/L of ADP cause irreversible platelet aggregation. Increases in transmural pressure induced endothelial depolarization in intact hamster small arterioles. Conclusions—Depolarization causes the endothelial production of O2-, which inhibits the activity of endothelial ectonucleotidases. Increases in transmural pressure induce endothelial depolarization. In chronically hypertensive diseases, depolarization might favor platelet aggregation.

Nutritionally Induced Obesity Is Attenuated in Transgenic Mice Overexpressing Plasminogen Activator Inhibitor-1 (Arteriosclerosis, Thrombosis, and Vascular Biology 2002) Objective—The objective of this study was to investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue development in vivo. Methods and Results—Transgenic (Tg) mice overexpressing murine PAI-1 under control of the adipocyte promoter aP2 and wild-type (WT) controls were kept on standard food (SFD) or on high-fat diet (HFD) for 15 weeks. The body weight and the weight of the isolated subcuttaneus and gonadal fat deposits of the Tg mice kept on the HFD were significantly lower than those of the WT mice. The number of adipocytes in the adipose tissue was similar for Tg and WT mice on the HFD, but adipocyte hypotrophy and a significantly lower ratio of stroma cells/adipocytes were observed in the Tg mice. A significant negative correlation (P<0.01) was observed between expression of preadipocyte factor-1, which blocks adipocyte differentiation, and adipose tissue weight. Fasting insulin and total cholesterol levels on the HFD were lower in Tg than in WT mice. Conclusions—High circulating PAI-1 levels attenuate nutritionally induced obesity. This may be related to modifications in adipose tissue cellularity affecting weight and plasma metabolic parameters.

• Increased apoA-I catabolism is found in dysmetabolic women and is consistent with the increased risk of atherosclerosis in this population. (Int J Obes Relat Metab Disord [2002] Sep;26(9):1151-8) Pont f , Duvillard l , Florentin e , Gambert p , Verges b AIMS/HYPOTHESIS: Mechanisms responsible for the decreased high-density lipoprotein (HDL) cholesterol level associated with insulin resistance in obese patients are not clearly understood. To determine the influence of insulin resistance at an early stage on HDL metabolism, we performed a stable isotope kinetic study of apolipoprotein (apo) A-I, in five obese insulin resistant women with normal fasting triglycerides and without impaired glucose tolerance, and in five age-matched control women. METHODS: Each subject received a 16 h constant infusion of L-[1-(13)C]leucine at 0.7 mg/kg/h following a primed bolus of 0.7 mg/kg. RESULTS: ApoA-I fractional catabolic rate (FCR) was significantly increased in insulin-resistantTan en compared to controls (0.316+/-0.056 vs 0.210+/-0.040 per day, P<0.01), indicating a significant 50% increase of apoA-I catabolism, leading to an important Tan ction of plasma apoA-I residence time (3.25+/-0.59 vs 4.92+/-1.11, P<0.01). ApoA-I production rate tended to be higher in insulin resistant wTan than in controls (364+/-77 vs 258+/-60 mg/l/day, P=0.13), but the difference was not statistically significant. ApoA-I FCR was correlated with triglycerides during the fed state (r=0.69; P=0.026) and HDL triglycerides-esterified cholesterol ratio (r=0.73; P=0.016), suggesting that alteration of apoA-I metabolism in insulin resistance mTan e partly related to HDL enrichment in triglycerides. CONCLUSIONS: Our kinetic study shows that patients, at an early stage of insulin resistance (without impaired glucose tolerance nor fasting hypertriglyceridaemia), already have a significant alteration of apoA-I metabolism (increased apoA-I catabolism), which is consistent with the increased risk of atherosclerosis in this population.

Serum homocysteine, creatinine, and glucose are predictors of the severity and extent of
coronary artery disease in asymptomatic members of high-risk families (Eur J Clin Invest [2002] 32:472) Pajunen P , Syvänne Mikko , Nieminen M S, Kareinen Anu , Viitanen Laura , Lehto Seppo , Laakso Markku Background There has been no previous study to determine the severity and extent of coronary artery disease (CAD) in subjects with no diagnosis or symptoms of CAD at the time of the angiography. Methods Fifty-three subjects, who were siblings of patients with early onset CAD, underwent coronary angiography. Indices to describe per-patient characteristics of CAD were calculated, based on computer-aided quantitative coronary angiography. Clinical and laboratory characteristics were correlated to the angiographic parameters. ResultsSerum total homocysteine ( = 0·29, P < 0·05) and creatinine ( = 0·47, P = 0·001) levels were related to the global atheroma burden index. The median of the atheroma burden index was two times higher in the top homocysteine quartile compared to the lowest quartile. The overall atheroma burden index correlated significantly with the fasting blood glucose level in all subjects. Diabetes, especially when albuminuria was present, was a powerful risk factor. In a multivariate analysis, only age and sex were independent predictors of atheroma burden. ConclusionsSerum homocysteine and creatinine concentrations, and diabetes with albuminuria were found to be markers of the severity and extent of CAD in subjects of high-risk families without symptoms of CAD.

• Unsaturated fatty acids and their oxidation products stimulate CD36 gene expression in human macrophages (Atherosclerosis [2002] 164:45-56) Fatty acids (FA) have been implicated in the control of expression of several atherosclerosis-related genes. Similarly, the CD36 receptor has recently been shown to play an important role in atherosclerosis and other pathologies. The aim of the present study was to evaluate the direct effect of FA and their oxidation products (aldehydes), on the expression of CD36 in both THP-1 macrophages and human monocyte-derived macrophages (HMDM). The FA tested included the saturated FA (SFA) lauric, myristic, palmitic and stearic acid; the monounsaturated FA oleic acid; and the unsaturated FA (UFA) linoleic, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Aldehydes used were malondialdehyde (MDA), hexanal, 2,4-decadienal (DDE) and 4-hydroxynonenal (HNE). CD36 expression was measured by RT-PCR, Western blot and immunofluorescence. Incubation of THP-1 macrophages for 24 h with non-cytotoxic concentrations of UFA significantly increased CD36 mRNA expression. By contrast, exposure of THP-1 macrophages to SFA did not affect the levels of CD36 mRNA. Among all UFAs tested, EPA and DHA were the strongest inducers of CD36 mRNA levels, followed by oleic and linoleic acid. Incubation of HMDM with either oleic or linoleic acid significantly increased steady-state CD36 mRNA in a dose-dependent manner. Consistent with the increase of CD36 mRNA expression, incubation of THP-1 macrophages with oleic and linoleic acid for 24 h markedly increased CD36 protein expression. Treatment of THP-1 macrophages with MDA or hexanal for 24 h significantly increased CD36 mRNA expression in a dose dependent manner. In contrast, DDE and HNE significantly decreased this parameter. The data provide evidence for a direct regulatory effect of UFA on CD36 gene expression and support a role for aldehydes in the regulation of CD36 expression by FA.

Fasting and postchallenge hyperglycaemia in the early phase of an acute myocardial infarction could be used as early markers of high-risk individuals (Lancet [2002] 359: 2140-44)Background Glycometabolic state at hospital admission is an important risk marker for long-term mortality in patients with acute myocardial infarction, whether or not they have known diabetes mellitus. Our aim was to ascertain the prevalence of impaired glucose metabolism in patients without diagnosed diabetes but with myocardial infarction, and to assess whether such abnormalities can be identified in the early course of a myocardial infarction. Methods We did a prospective study, in which we enrolled 181 consecutive patients admitted to the coronary care units of two hospitals in Sweden with acute myocardial infarction, no diagnosis of diabetes, and a blood glucose concentration of less than 11·1 mmol/L. We recorded glucose concentrations during the hospital stay, and did standardised oral glucose tolerance tests with 75 g of glucose at discharge and again 3 months later. Findings The mean age of our cohort was 63·5 years (SD 9) and the mean blood glucose concentration at admission was 6·5 mmol/L (1·4). The mean 2-h postload blood glucose concentration was 9·2 mmol/L (2·9) at hospital discharge, and 9·0 mmol/L (3·0) 3 months later. 58 of 164 (35%, 95% CI 28-43) and 58 of 144 (40%, 32-48) individuals had impaired glucose tolerance at discharge and after 3 months, respectively, and 51 of 164 (31%, 24-38) and 36 of 144 (25%, 18-32) had previously undiagnosed diabetes mellitus. Independent predictors of abnormal glucose tolerance at 3 months were concentrations of HbA_1c at admission (p=0·024) and fasting blood glucose concentrations on day 4 (p=0·044). Interpretation Previously undiagnosed diabetes and impaired glucose tolerance are common in patients with an acute myocardial infarction. These abnormalities can be detected early in the postinfarction period. Our results suggest that fasting and postchallenge hyperglycaemia in the early phase of an acute myocardial infarction could be used as early markers of high-risk individuals.

• Interleukin-18 Is a Strong Predictor of Cardiovascular Death in Stable and Unstable Angina (Circulation [2002]106:24) Background— Interleukin (IL)-18 plays a central role in orchestrating the cytokine cascade and accelerates atherosclerosis and plaque vulnerability in animal models. However, epidemiological data evaluating the role of IL-18 levels in atherosclerosis are lacking. M ethods and Results— In a prospective study of 1229 patients with documented coronary artery disease, we measured baselinemarkers of serum concentrations of IL-18 and other inflammation. During the follow-up period (median, 3.9 years), 95 patients died of cardiovascular causes. Median serum concentrations of IL-18 were significantly higher among patients who had a fatal cardiovascular event than among those who did not (68.4 versus 58.7 pg/mL; P<0.0001). The hazard risk ratio of future cardiovascular death increased with increasing quartiles of IL-18 (hazard risk ratio, 1.46; 95% CI 1.21 to 1.76; P for trend <0.0001). After adjustment for most potential confounders, including the strong predictor ejection fraction as well as the inflammatory variables IL-6, high-sensitive C-reactive protein, and fibrinogen, this relation remained almost unchanged, such that patients within the highest quartile of IL-18 had a 3.3-fold increase in hazard risk compared with those in the first quartile (95% CI, 1.3 to 8.4, P=0.01). This relation was observed in patients with stable angina and patients with unstable angina at baseline. Conclusions— Serum IL-18 level was identified as a strong independent predictor of death from cardiovascular causes in patients with coronary artery disease regardless of the clinical status at admission. This result strongly supports recent experimental evidence of IL-18–mediated inflammation leading to acceleration and vulnerability of atherosclerotic plaques.

Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway. (Circulation [2002] 105:2198.)Background— Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known. Methods and Results— To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10^-7 mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT₁R), insofar as an AT₁R antagonist (AT₁a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT₂R) antagonist (AT₂a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-kappaB and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-kappa-B-alpha activation was accompanied by phosphorylation of Ikappa B^alpha on serine 32 as well as degradation of IkappaB-alpha, suggesting that the effects of Ang II were mediated through an IkappaBalpha-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II–induced TNF biosynthesis. Conclusions— These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.

• Proinsulin Is an Independent Predictor of Coronary Heart Disease (Circulation [2002] 105:2153) Background— Some, but not all, studies have reported a relationship between plasma insulin and coronary heart disease (CHD). Conventional nonspecific insulin assays are also measuring various fractions of proinsulin-like molecules due to cross-reactivity. The long-term relationship between proinsulin-like molecules and CHD is largely unknown. For this reason, the longitudinal relationships between intact proinsulin, split proinsulin, specific insulin, immunoreactive insulin, and CHD, were studied in a population-based cohort of 50-year-old men (n=874), with a follow-up of 27 years. Methods and Results— Fasting proinsulin-like molecule and specific-insulin concentrations were measured in plasma (stored frozen since baseline 1970 to 1973) by specific 2-site immunometric assays. Immunoreactive insulin concentrations were determined at baseline. The associations between proinsulin-like molecules, specific insulin, immunoreactive insulin, and CHD mortality (International Classification of Diseases [9th revision] codes 410 to 414) were analyzed using Cox’s proportional hazards regression and presented as hazard ratios (HRs) with their 95% confidence intervals (CIs) for a 1-SD increase in a predictor variable. In the univariate analysis, intact proinsulin (HR, 1.69; 95% CI, 1.41 to 2.01) was the strongest predictor of death from CHD. In the multivariate analysis, smoking (HR, 1.57; 95% CI, 1.03 to 2.38), intact proinsulin (HR, 1.47; 95% CI, 1.18 to 1.82), systolic blood pressure (HR, 1.38; 95% CI, 1.14 to 1.66), and LDL/HDL cholesterol ratio (HR, 1.31; 95% CI, 1.12 to 1.53) were independent predictors of CHD mortality (adjusted for body mass index, triglycerides, and fasting glucose), whereas specific insulin and immunoreactive insulin were not (HR, 1.12; 95% CI, 0.90 to 1.40). The increased risk was restricted to the upper third of the proinsulin distribution. Conclusion— Increased proinsulin concentrations predict death and morbidity caused by CHD over a period of 27 years, independent of other major cardiovascular risk factors

Atrial Natriuretic Peptide Reduces TNF-Induced Actin Polymerization and Endothelial Permeability (Circulation Research [2002]90:874) The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-alpha on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-alpha lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10(-8) to 10(-6) mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-alpha-induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-alpha-induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-alpha-induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1-induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.

• The Kir6.1-containing K+ channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina. (Nature Med [2002] 8:466-472) The inwardly rectifying K⁺ channel Kir6.1 forms K⁺ channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K⁺ channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K⁺ channel opener pinacidil did not induce K⁺ currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K⁺ channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.

Insulin resistance in moderate chronic heart failure is related to hyperleptinaemia, but not to norepinephrine or TNF-alpha (International Journal of Cardiology [2002] 83:73-81) Objectives: Chronic heart failure (CHF) has emerged as an insulin-resistant state, independently of ischaemic aetiology. The underlying mechanisms of this finding are not known. Catecholamines, tumor necrosis factor alpha (TNF) and leptin, the adipocyte specific hormone, have all been implicated as mediators of impaired insulin sensitivity. The purpose of this study was to examine in patients with CHF and in comparison to healthy controls subjects whether norepinephrine, TNF or leptin relate to insulin sensitivity. Design: 41 patients with CHF (age 60±2 years, NYHA I/II/III/IV 4/12/22/3, peak oxygen consumption 17.6±1.0 ml/kg per min) and 21 healthy controls of similar age and total and regional fat distribution were studied in a cross-sectional study. Insulin sensitivity was assessed by intravenous glucose tolerance testing using the minimal model approach; catecholamines, TNF and soluble TNF receptors 1 and 2 were also measured. Total and regional body fat mass was assessed by dual energy X-ray absorptiometry. Results: Insulin sensitivity was reduced in CHF patients compared to controls by 31% (P<0.01) and fasting insulin was higher in patients than in controls (79.1±9.7 vs. 41.4±6.0 pmol/l, P<0.01). Patients had, compared to healthy controls, elevated serum leptin levels (8.28±0.84 vs. 4.83±0.68 ng/ml), norepinephrine (3.45±0.34 vs. 1.87±0.16 nmol/l, both P<0.01) and soluble TNF-receptors 1 (1280±141 vs. 639±52 pg/ml) and 2 (2605±184 vs. 1758±221 pg/ml, both P<0.01). Leptin levels corrected for total body fat mass were higher in CHF patients than in controls (41.3±3 vs. 24.3±2 pg/ml per 100 g, P<0.001). TNF was not significantly different between the groups. In both groups there was an inverse correlation between insulin sensitivity and serum leptin (r=-0.65, P<0.0001 for pooled subjects); in contrast, no significant relation was found between insulin sensitivity and norepinephrine or TNF. In multivariate regression analysis, leptin emerged as the only significant predictor of insulin sensitivity (standardised COEFFICIENT=-0.59, P<0.001), independent of body fat mass, age and peak V₂. Conclusion: In moderate CHF, elevated leptin levels directly and independently predict insulin resistance. Elevated serum leptin levels could play a role in the impaired regulation of energy metabolism in CHF. In contrast to observations in other conditions, TNF and norepinephrine are not related to insulin resistance in moderate CHF.

• Leptin contributes to arterial thrombosis following vascular injury in vivo (JAMA. [2002] 287:1706-1709) Context  Complications of atherosclerosis are the leading cause of morbidity and mortality in industrialized societies. Obesity has emerged as an independent risk factor for complications of atherosclerotic vascular disease. Leptin, a hormone produced by the adipocyte, increases with obesity and appears to modulate energy balance and food intake. In addition, other actions of leptin have been proposed, including an in vitro effect on platelet aggregation. Thus, the elevated plasma leptin levels in obese individuals may promote vascular thrombosis. Objective  To test the hypothesis that leptin contributes to in vivo thrombosis via the leptin receptor. Design and Materials  Between September 2000 and September 2001, a vascular thrombosis model was used to test male 10- to 12-week-old mice completely deficient in leptin or the leptin receptor and mice with platelet leptin-receptor deficiency. Main Outcome Measure  Time to formation of an occlusive thrombus in the common carotid artery following experimentally induced endothelial injury. Results  Following onset of vascular injury, wild-type mice (n = 8) formed occlusive thrombosis in a mean (SD) of 42.2 (4.6) minutes, whereas leptin-deficient (n = 5) and leptin receptor–deficient mice (n = 7) formed occlusive thrombosis in 75.2 (10.1) and 68.6 (10.3) minutes, respectively (leptin deficient vs wild-type mice, P = .008; leptin-receptor–deficient vs wild-type, P = .03). When recombinant murine leptin was administered to leptin-deficient mice (n = 4), the time to occlusion was reduced to 41.8 (6.6) minutes (P = .035 vs vehicle control). Following bone marrow transplantation from leptin receptor–deficient (donor) mice to wild-type (recipient) mice, the time to occlusion wa