Charalambos Antoniades

Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials

Plasma total homocysteine (Hcy) has been associated with cardiovascular risk in multiple large-scale epidemiological studies, and it has been considered as an independent risk factor for atherosclerosis. Homocysteine lowering, achieved after the introduction of the folate food fortification programme in North America, was accompanied by an accelerated decline of cardiovascular risk and especially of stroke. Although the initial clinical trials suggested that homocysteine-lowering treatment with folates and B vitamins induces coronary plaque regression, this finding was not confirmed by more recent clinical studies. Under the light of the findings from the recent large randomized clinical trials that failed to document a benefit of Hcy lowering on clinical outcome of patients with atherosclerosis, the role of Hcy as a risk factor and the efficacy of Hcy lowering against atherosclerosis have been questioned. Therefore, better understanding of the mechanisms relating Hcy and Hcy-lowering treatment with vascular function and atherogenesis is crucial, to help us understand why clinical trials failed to show a benefit from Hcy-lowering treatment. Are these therapeutic strategies ineffective because they fail to reduce intracellular Hcy levels and vascular redox state or should Hcy stop being considered as an independent risk factor for atherosclerosis from now on? In this review article, we provide a global approach of the molecular mechanisms relating Hcy with cardiovascular risk and introduce possible mechanistic explanations regarding the inability of clinical trials to detect any clinical benefit from Hcy-lowering treatment in secondary prevention. Finally, we provide clinical recommendations regarding the therapeutic strategies targeting homocysteine in the general population.

Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis

BACKGROUND Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear. AIMS We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advanced atherosclerosis. METHODS AND RESULTS Paired samples of saphenous veins (SVs) and internal mammary arteries (IMAs) were collected from 201 patients undergoing coronary bypass surgery, and serum ADMA was measured pre-operatively. The vasomotor responses of SV segments to acetylcholine (ACh) and bradykinin (Bk) were evaluated ex vivo. Vascular O2- was measured in paired SV and IMA by lucigenin-enhanced chemiluminescence. The l-NAME-inhibitable as well as the NADPH-stimulated vascular O2- generation was also determined by chemiluminescence. High serum ADMA levels were associated with decreased vasorelaxation of SV to ACh (P < 0.05) and Bk (P < 0.05). Similarly, high serum ADMA was associated with higher total O2- production in both SVs and IMAs (P < 0.05) and greater L-NAME-inhibitable vascular O2- (P < 0.05). However, serum ADMA was not associated with NADPH-stimulated vascular O2-. In multivariable linear regression, serum ADMA was independently associated with vascular O2- in both SVs [beta (SE): 0.987 (0.412), P = 0.019] and IMAs [beta (SE): 1.905 (0.541), P = 0.001]. Asymmetrical dimethylarginine was also independently associated with maximum vasorelaxation in response to both ACh [beta (SE): 14.252 (3.976), P = 0.001] and Bk [beta (SE): 9.564 (3.762), P = 0.013]. CONCLUSION This is the first study that demonstrates an association between ADMA and important measures of vascular function, such as vascular O2- production and NO bioavailability directly in human vessels. Although serum ADMA has no effect on NADPH-stimulated superoxide in intact vessels, it is associated with greater eNOS uncoupling in the human vascular endothelium of patients with coronary artery disease.

Adiponectin: from obesity to cardiovascular disease

Adiponectin is an adipokine whose biosynthesis is deranged in obesity and diabetes mellitus, predisposing to atherosclerosis. Evidence suggests that adiponectin has anti‐atherogenic properties by improving endothelial function and having anti‐inflammatory effects in the vascular wall. In addition, adiponectin modifies vascular intracellular redox signalling and exerts indirect antioxidant effects on human myocardium. However, its clinical role in cardiovascular disease is obscure. Adiponectins positive prognostic value in coronary artery disease had been widely supported over the last years, but this view has been questioned recently. High adiponectin levels are paradoxically associated with poorer prognosis in heart failure syndrome. These controversial findings seem surprising as adiponectin has been viewed overall as an anti‐atherogenic molecule. Therefore, any certain conclusion about adiponectins role in cardiovascular disease seems premature. Despite the rapidly accumulating literature on this adipokine, it is still unclear whether adiponectin is a key mediator or a bystander in cardiovascular disease. It is still uncertain whether adiponectin levels have any clinical significance for risk stratification in cardiovascular disease or they just reflect the activation of complex and opposing underlying mechanisms. Circulating adiponectin levels should be interpreted with caution, as they may have completely different prognostic value, depending on the underlying disease state.

Evaluating endothelial function in humans: a guide to invasive and non-invasive techniques

The endothelium regulates vascular homeostasis through the release of a variety of autocrine and paracrine substances, such as nitric oxide (NO), prostacyclin, and the endothelium derived hyperpolarising factor(s).1 Beyond its vasodilatory effects, NO has antiatherogenic properties, inhibits platelet aggregation and adhesion, smooth muscle cell proliferation, leucocyte adhesion, vascular permeability, and inflammatory mechanisms (fig 1).1 Figure 1  Effects of different stimuli (physical or chemical) on vascular endothelium. Stimuli leading to vasorelaxation in the presence of intact vascular endothelium (such as acetylcholine), produce vasoconstriction when acting directly on the underlying smooth muscle cells, in vascular areas with injured endothelium. CPT, cold pressor stress test; EDHF, endothelium derived hyperpolarising factor; eNOS, endothelial nitric oxide synthase; GC, guanyl cyclase; L-NMMA, NG-monomethyl-L-arginine; MS, mental stress; NO, nitric oxide; SMC, smooth muscle cell. Several prospective and retrospective studies have shown that evaluation of endothelial function may be useful in identifying subjects at high risk for atherosclerosis, as well as for risk stratification of patients with already established coronary artery disease.2w1 Several invasive and non-invasive techniques have been developed during the last few years to evaluate endothelial function. Invasive techniques, which involve intracoronary or intrabrachial infusions of vasoacting agents, are still considered to be the gold standard for early detection of endothelial dysfunction. In addition, several non-invasive techniques have been developed, with comparable results and good reproducibility. In this article we describe and evaluate the different methods for assessing endothelial function in humans. ### Intracoronary infusions of vasoactive agents Intracoronary agonist infusion, combined with quantitative angiography, is the method of choice for direct quantification of endothelial function in the coronary arteries, since it allows both the evaluation of dose–response relations of endothelial agonists and antagonists, as well as assessing the basal endothelial function by the infusion of nitric oxide synthase (NOS) inhibitors.2 It is well known that intracoronary …

Role of inflammation and oxidative stress in endothelial progenitor cell function and mobilization: Therapeutic implications for cardiovascular diseases

Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the peripheral circulation, home to sites of injury, and incorporate into foci of neovascularization, thereby improving blood flow and tissue recovery. Patients with cardiovascular diseases, including coronary artery disease, heart failure, hypertension, and diabetes, have been shown to exhibit reduced number and functional capacity of EPCs. Considerable evidence indicates that EPCs constitute an important endogenous system to maintain endothelial integrity and vascular homeostasis, while reduced number of EPCs has recently been shown to predict future cardiovascular events. Thus, enhancement of EPCs could be of potential benefit for individuals with cardiovascular diseases. The interplay between inflammation and oxidative stress is involved in the initiation, progression, and complications of cardiovascular diseases. Emerging evidence from in vitro and clinical studies suggests that inflammatory and oxidative changes influence EPC mobilization. Drugs with anti-inflammatory and antioxidant properties, currently administered to patients with cardiovascular diseases, such as statins, have been demonstrated to exert beneficial effects on EPC biology. A better understanding of the inflammatory and oxidative mechanisms leading to the numerical and functional impairment of EPCs would provide additional insight into the pathogenesis of cardiovascular disease and create novel therapeutic targets.

Effects of combined administration of low dose atorvastatin and vitamin E on inflammatory markers and endothelial function in patients with heart failure.

Heart failure has been associated with impaired endothelial function, increased inflammatory process and elevated oxidative stress status. Both statins and vitamin E separately improve endothelial function in patients with hypercholesterolemia and/or advanced atherosclerosis.

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).

Biomarkers of premature atherosclerosis

C-reactive protein (CRP) is an acute phase protein and a biochemical marker with important prognostic value for cardiovascular events. Interleukins IL-1 and IL-6 are implicated in the pathogenesis of atherosclerosis and are associated with CRP. Apolipoproteins ApoA-I and ApoB are the main lipid metabolic markers implicated in the development and progression of atherosclerosis. Fibrinogen has also been proposed to be a major independent risk factor for cardiovascular events. Because premature atherosclerosis precedes the development of cardiovascular disease, identification of the associated biomarkers is of great importance. However, further studies will be needed to determine whether or not these markers are useful predictors of future cardiovascular events. Here, we review the roles of specific biomarkers that have been implicated in premature atherosclerosis.

Effects of Ramipril on Endothelial Function and the Expression of Proinflammatory Cytokines and Adhesion Molecules in Young Normotensive Subjects With Successfully Repaired Coarctation of Aorta: A Randomized Cross-Over Study

OBJECTIVES The purpose of this study was to evaluate the effect of ramipril on endothelial function and inflammatory process in a group of normotensive subjects with successfully repaired coarctation of the aorta (SCR). BACKGROUND Subjects with SCR experience higher long-term cardiovascular risk as a result of the relapse of arterial hypertension or owing to nonreversible structural changes in the pre-coarctation arterial tree. These subjects experience endothelial dysfunction in the right forearm and appear to have elevated levels of proatherogenic inflammatory markers, even in the absence of arterial hypertension. METHODS Twenty young individuals age 27.3 +/- 2.4 years old with SCR 13.9 +/- 2.2 years previously, received ramipril 5 mg/day for 4 weeks in a randomized, cross-over, controlled trial. Endothelial function was evaluated in the right forearm by gauge-strain plethysmography, and serum levels of interleukin (IL)-1b, IL-6, soluble CD40 ligand (sCD40L), and soluble vascular cell adhesion molecule (sVCAM)-1 were determined by enzyme-linked immunosorbent assay. RESULTS Ramipril improved endothelial function (p < 0.001) and decreased the expression of proinflammatory cytokine IL-6 (p < 0.05) and sCD40L (p < 0.01). Furthermore, ramipril decreased serum levels of sVCAM-1 (p < 0.01) but failed to affect serum levels of C-reactive protein. These effects were independent of blood pressure lowering. CONCLUSIONS Ramipril reversed the impaired endothelial function and decreased the expression of proinflammatory cytokine IL-6, sCD40L, and adhesion molecules in normotensive subjects with SCR. These findings imply that ramipril treatment may have antiatherogenic effects in subjects with SCR, even in the absence of arterial hypertension.

Assessing inflammatory status in cardiovascular disease

Growing evidence suggests that the immune system plays a critical role in all the stages of atherogenesis, from lesion formation to plaque rupture.1 The inflammatory component of atherothrombosis is a topic of intensive research, since it is still unclear whether inflammatory markers measurable in peripheral blood can be useful tools for risk assessment in the general population; it is also unknown whether therapeutic strategies leading to a decrease of these inflammatory markers can modify cardiovascular risk.w1 Early in the atherogenesis process, resident or circulating leucocytes bind to the site of a developing lesion in response to oxidised low density lipoprotein cholesterol (LDL-C), injury, or infection. Proinflammatory cytokines play a central regulatory role in this early stage of atherogenesis, since they induce the migration of these inflammatory cells to the subendothelial space, both by acting directly on these leucocytes and by upregulating the expression of several adhesion molecules (such as vascular cells adhesion molecules, intercellular adhesion molecules and selectins) which participate in leucocyte adhesion, rolling and subendothelial migration (fig 1).w2 Figure 1  Cytokines and adhesion molecules in the early stages of atherogenesis. Circulating leucocytes are activated by proinflammatory cytokines, and they adhere to vascular endothelium. Adhesion molecules, whose expression is also regulated by proinflammatory cytokines, play a key role in the subendothelial migration of leucocytes. ICAM-1, intercellular adhesion molecule-1; PECAM-1, platelet endothelial cell adhesion molecule; Sel, selectin; VCAM-1, vascular cell adhesion molecule-1. As these monocytes accumulate in the subendothelial space, they continue to ingest chemically modified lipids and lipoproteins, they become macrophages and finally develop into foam cells, and initiate fatty streaks. At the same time, other inflammatory cells, such as activated T cells and mast cells, also attach themselves to the endothelium, contributing to the formation of the atheromatous lesion, which consists of a lipid pool covered by a …