Marios Margaritis

Statins as Anti-Inflammatory Agents in Atherogenesis: Molecular Mechanisms and Lessons from the Recent Clinical Trials

Ample evidence exists in support of the potent anti-inflammatory properties of statins. In cell studies and animal models statins exert beneficial cardiovascular effects. By inhibiting intracellular isoprenoids formation, statins suppress vascular and myocardial inflammation, favorably modulate vascular and myocardial redox state and improve nitric oxide bioavailability. Randomized clinical trials have demonstrated that further to their lipid lowering effects, statins are useful in the primary and secondary prevention of coronary heart disease (CHD) due to their anti-inflammatory potential. The landmark JUPITER trial suggested that in subjects without CHD, suppression of low-grade inflammation by statins improves clinical outcome. However, recent trials have failed to document any clinical benefit with statins in high risk groups, such in heart failure or chronic kidney disease patients. In this review, we aim to summarize the existing evidence on statins as an anti-inflammatory agent in atherogenesis. We describe the molecular mechanisms responsible for the anti-inflammatory effects of statins, as well as clinical data on the non lipid-lowering, anti-inflammatory effects of statins on cardiovascular outcomes. Lastly, the controversy of the recent large randomized clinical trials and the issue of statin withdrawal are also discussed.

Evaluating Oxidative Stress in Human Cardiovascular Disease: Methodological Aspects and Considerations

Oxidative stress is a key feature in atherogenesis, since reactive oxygen species (ROS) are involved in all stages of the disease, from endothelial dysfunction to atheromatic plaque formation and rupture. It is therefore important to identify reliable biomarkers allowing us to monitor vascular oxidative stress status. These may lead to improved understanding of disease pathogenesis and development of new therapeutic strategies. Measurement of circulating biomarkers of oxidative stress is challenging, since circulation usually behaves as a separate compartment to the individual structures of the vascular wall. However, measurement of stable products released by the reaction of ROS and vascular/circulating molecular structures is a particularly popular approach. Serum lipid hydroperoxides, plasma malondialdehyde or urine F2-isoprostanes are widely used and have a prognostic value in cardiovascular disease. Quantification of oxidative stress at a tissue level is much more accurate. Various chemiluminescence and high performance liquid chromatography assays have been developed over the last few years, and some of them are extremely accurate and specific. Electron spin resonance spectroscopy and micro-electrode assays able to detect ROS directly are also widely used. In conclusion, measurement of circulating biomarkers of oxidative stress is valuable, and some of them appear to have predictive value in cardiovascular disease. However, these biomarkers do not necessarily reflect intravascular oxidative stress and therefore cannot be used as therapeutic targets or markers to monitor pharmacological treatments in clinical settings. Measurement of vascular oxidative stress status is still the only reliable way to evaluate the involvement of oxidative stress in atherogenesis.

Interactions Between Vascular Wall and Perivascular Adipose Tissue Reveal Novel Roles for Adiponectin in the Regulation of Endothelial Nitric Oxide Synthase Function in Human Vessels

Background— Adiponectin is an adipokine with potentially important roles in human cardiovascular disease states. We studied the role of adiponectin in the cross-talk between adipose tissue and vascular redox state in patients with atherosclerosis. Methods and Results— The study included 677 patients undergoing coronary artery bypass graft surgery. Endothelial function was evaluated by flow-mediated dilation of the brachial artery in vivo and by vasomotor studies in saphenous vein segments ex vivo. Vascular superoxide (O2−) and endothelial nitric oxide synthase (eNOS) uncoupling were quantified in saphenous vein and internal mammary artery segments. Local adiponectin gene expression and ex vivo release were quantified in perivascular (saphenous vein and internal mammary artery) subcutaneous and mesothoracic adipose tissue from 248 patients. Circulating adiponectin was independently associated with nitric oxide bioavailability and O2− production/eNOS uncoupling in both arteries and veins. These findings were supported by a similar association between functional polymorphisms in the adiponectin gene and vascular redox state. In contrast, local adiponectin gene expression/release in perivascular adipose tissue was positively correlated with O2− and eNOS uncoupling in the underlying vessels. In ex vivo experiments with human saphenous veins and internal mammary arteries, adiponectin induced Akt-mediated eNOS phosphorylation and increased tetrahydrobiopterin bioavailability, improving eNOS coupling. In ex vivo experiments with human saphenous veins/internal mammary arteries and adipose tissue, we demonstrated that peroxidation products produced in the vascular wall (ie, 4-hydroxynonenal) upregulate adiponectin gene expression in perivascular adipose tissue via a peroxisome proliferator-activated receptor-&ggr;–dependent mechanism. Conclusions— We demonstrate for the first time that adiponectin improves the redox state in human vessels by restoring eNOS coupling, and we identify a novel role of vascular oxidative stress in the regulation of adiponectin expression in human perivascular adipose tissue.

Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients With Coronary Artery Disease

Background— The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease. Methods and Results— Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue. Conclusions— Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00423280.

Adiponectin as a Link Between Type 2 Diabetes and Vascular NADPH Oxidase Activity in the Human Arterial Wall: The Regulatory Role of Perivascular Adipose Tissue

Oxidative stress plays a critical role in the vascular complications of type 2 diabetes. We examined the effect of type 2 diabetes on NADPH oxidase in human vessels and explored the mechanisms of this interaction. Segments of internal mammary arteries (IMAs) with their perivascular adipose tissue (PVAT) and thoracic adipose tissue were obtained from 386 patients undergoing coronary bypass surgery (127 with type 2 diabetes). Type 2 diabetes was strongly correlated with hypoadiponectinemia and increased vascular NADPH oxidase–derived superoxide anions (O2˙−). The genetic variability of the ADIPOQ gene and circulating adiponectin (but not interleukin-6) were independent predictors of NADPH oxidase–derived O2˙−. However, adiponectin expression in PVAT was positively correlated with vascular NADPH oxidase–derived O2˙−. Recombinant adiponectin directly inhibited NADPH oxidase in human arteries ex vivo by preventing the activation/membrane translocation of Rac1 and downregulating p22phox through a phosphoinositide 3-kinase/Akt-mediated mechanism. In ex vivo coincubation models of IMA/PVAT, the activation of arterial NADPH oxidase triggered a peroxisome proliferator–activated receptor-γ–mediated upregulation of the adiponectin gene in the neighboring PVAT via the release of vascular oxidation products. We demonstrate for the first time in humans that reduced adiponectin levels in individuals with type 2 diabetes stimulates vascular NADPH oxidase, while PVAT “senses” the increased NADPH oxidase activity in the underlying vessel and responds by upregulating adiponectin gene expression. This PVAT-vessel interaction is identified as a novel therapeutic target for the prevention of vascular complications of type 2 diabetes.

Myocardial Redox State Predicts In-Hospital Clinical Outcome After Cardiac Surgery Effects of Short-Term Pre-Operative Statin Treatment

OBJECTIVES The purpose of this study was to evaluate the role of the myocardial redox state in the development of in-hospital complications after cardiac surgery and the effect of statins on the myocardial redox state. BACKGROUND Statins improve clinical outcome after cardiac surgery, but it is unclear whether they exert their effects by modifying the myocardial redox state. METHODS We quantified myocardial superoxide anion (O(2)(-)) and peroxynitrite (ONOO(-)) and their enzymatic sources in samples of the right atrial appendage (RAA) from 303 patients undergoing cardiac surgery who were followed up until discharge, and in 42 patients who were randomized to receive 3-day treatment with atorvastatin 40 mg/d or placebo before surgery. The mechanisms by which atorvastatin modifies myocardial redox state were investigated in 26 RAA samples that were exposed to atorvastatin ex vivo. RESULTS Atrial O(2)(-) (derived mainly from nicotinamide adenine dinucleotide phosphate [NADPH] oxidases) and ONOO(-) were independently associated with increased risk of atrial fibrillation, the need for post-operative inotropic support, and the length of hospital stay. Pre-operative atorvastatin treatment suppressed atrial NADPH oxidase activity and myocardial O(2)(-) and ONOO(-) production. Ex vivo incubation of RAA samples with atorvastatin induced a mevalonate-reversible and Rac1-mediated inhibition of NADPH oxidase. CONCLUSIONS There is a strong independent association between myocardial O(2)(-)/ONOO(-) and in-hospital complications after cardiac surgery. Both myocardial O(2)(-) and ONOO(-) are reduced by pre-operative statin treatment, through a Rac1-mediated suppression of NADPH oxidase activity. These findings suggest that inhibition of myocardial NADPH oxidases may contribute to the beneficial effect of statins in patients undergoing cardiac surgery. (Effects of Atorvastatin on Endothelial Function, Vascular and Myocardial Redox State in High Cardiovascular Risk Patients; NCT01013103).

Statins as Regulators of Redox State in the Vascular Endothelium: Beyond Lipid Lowering

SIGNIFICANCE Endothelial dysfunction and the imbalance between nitric oxide (NO) and reactive oxygen species production in the vascular endothelium are important early steps in atherogenesis, a major socioeconomic health problem. Statins have well-established roles in primary and secondary prevention of cardiovascular disease (CVD), due to both their lipid-lowering capacity and their pleiotropic properties. It is therefore important to understand the mechanisms by which statins can modify endothelial function and affect atherogenesis. RECENT ADVANCES In the last decade, the concept of statin pleiotropy has been reinforced by a large number of cell culture, animal, and translational studies. Statins have been shown to suppress the activity of pro-oxidant enzymes (such as NADPH oxidase) and pro-inflammatory transcriptional pathways in the endothelium. At the same time, they enhance endothelial NO synthase expression and activity while they also improve its enzymatic coupling. This leads to increased NO bioavailability and improved endothelial function. CRITICAL ISSUES Despite significant recent advances, the exact mechanisms of statin pleitropy are still only partially understood. The vast majority of the published literature relies on animal studies, while the actual mechanistic studies in humans are limited. FUTURE DIRECTIONS The success of statins as endothelium redox-modifying agents with a direct impact on clinical outcome highlights the importance of the endothelium as a therapeutic target in CVD. Better understanding of the mechanisms that underlie endothelial dysfunction could lead to the design of novel therapeutic strategies that target the vascular endothelium for the prevention and treatment of CVD.

Nanomedicine for the prevention, treatment and imaging of atherosclerosis.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in developed countries, with an increasing prevalence due to an aging population. The pathology underpinning CVD is atherosclerosis, a chronic inflammatory state involving the arterial wall. Accumulation of low density lipoprotein (LDL) laden macrophages in the arterial wall and their subsequent transformation into foam cells lead to atherosclerotic plaque formation. Progression of atherosclerotic lesions may gradually lead to plaque related complications and clinically manifest as acute vascular syndromes including acute myocardial or cerebral ischemia. Nanotechnology offers emerging therapeutic strategies, which may have advantage overclassical treatments for atherosclerosis. In this review, we present the potential applications of nanotechnology toward prevention, identification and treatment of atherosclerosis.

Induction of Vascular GTP-Cyclohydrolase I and Endogenous Tetrahydrobiopterin Synthesis Protect Against Inflammation-Induced Endothelial Dysfunction in Human Atherosclerosis

Background— The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. Methods and Results— In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-&agr;/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. Conclusions— The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Reciprocal Effects of Systemic Inflammation and Brain Natriuretic Peptide on Adiponectin Biosynthesis in Adipose Tissue of Patients With Ischemic Heart Disease

Objective— To explore the role of systemic inflammation in the regulation of adiponectin levels in patients with ischemic heart disease. Approach and Results— In a cross-sectional study of 575 subjects, serum adiponectin was compared between healthy subjects, patients with coronary artery disease with no/mild/severe heart failure (HF), and patients with nonischemic HF. Adiponectin expression and release from femoral, subcutaneous and thoracic adipose tissue was determined in 258 additional patients with coronary artery bypass grafting. Responsiveness of the various human adipose tissue depots to interleukin-6, tumor necrosis factor-&agr;, and brain natriuretic peptide (BNP) was examined by using ex vivo models of human fat. The effects of inducible low-grade inflammation were tested by using the model of Salmonella typhi vaccine-induced inflammation in healthy individuals. In the cross-sectional study, HF strikingly increased adiponectin levels. Plasma BNP was the strongest predictor of circulating adiponectin and its release from all adipose tissue depots in patients with coronary artery bypass grafting, even in the absence of HF. Femoral AT was the depot with the least macrophages infiltration and the largest adipocyte cell size and the only responsive to systemic and ex vivo proinflammatory stimulation (effect reversible by BNP). Low-grade inflammation reduced circulating adiponectin levels, while circulating BNP remained unchanged. Conclusions— This study demonstrates the regional variability in the responsiveness of human adipose tissue to systemic inflammation and suggests that BNP (not systemic inflammation) is the main driver of circulating adiponectin in patients with advanced atherosclerosis even in the absence of HF. Any interpretation of circulating adiponectin as a biomarker should take into account the underlying disease state, background inflammation, and BNP levels.