Clinical utility of biomarkers in premature atherosclerosis.

Atherosclerosis is a very complex procedure responsible for the development of coronary artery disease which is the leading cause of death in the civilized world. The obvious pandemic character of atherosclerosis augments the need to discover an ideal biomarker, which will be able to facilitate the clinical diagnosis of the atherosclerosis from the physicians especially in the early stages of the atherosclerotic process. Among the biomarkers that are already used there are classical ones, such as c-reactive protein, interleukins, tumour necrosis factor, apolipoproteins, fibrinogen, homocysteine, and novel promising ones such as lipoprotein-associated phospholipase, asymmetric dimethylarginine, myeloperoxidase, cathepsins and cystatin C. The possibility of combining circulating biomarkers with other methods such as non-invasive and invasive imaging is clinically attractive because this could contribute to the improved diagnosis and understanding of premature atherosclerosis pathogenesis.

The role of microRNAs in cardiovascular disease.

Cardiovascular disease, which is multifactorial and can be influenced by a multitude of environmental and heritable risk factors, remains a major health problem, even though its pathophysiology is far from been elucidated. Discovered just over a decade ago, microRNAs comprise short, non-coding RNAs, which have evoked a great deal of interest, due to their importance for many aspects of homeostasis and disease. Hundreds of different microRNAs are constantly being reported in various organisms. According to a growing body of literature, they have been implicated in the regulation of human physiological processes. More specifically, miRNAs are expressed in the cardiovascular system and could have crucial roles in normal development and physiology, as well as in disease development. Furthermore, they have been shown to participate in cardiovascular disease pathogenesis including atherosclerosis, coronary artery disease, myocardial infarction, heart failure and cardiac arrhythmias. In contrast to our original thought, miRNAs exist in circulating blood and are relatively stable, thus, they could be proved useful as biomarkers in that state. Understanding the underlying mechanisms, in which these major regulatory gene families are implicated, will provide novel opportunities for diagnosis and therapy of cardiovascular diseases.

Effects of vitamin C on intracoronary L-arginine dependent coronary vasodilatation in patients with stable angina.

OBJECTIVE: To assess the effects of intravenous vitamin C administration on the vasomotor responses to intracoronary L-arginine infusion in epicardial coronary arteries. METHODS: 28 patients with coronary artery disease and stable angina were enrolled in the study. Eight patients received intracoronary infusions of 150 micromol/min L-arginine before and after intravenous infusion of vitamin C, 10 patients received intracoronary infusions of 150 micromol/min L-arginine before and after intravenous infusion of normal saline, and 10 patients received intracoronary normal saline before and after intravenous infusion of vitamin C. The diameter of proximal and distal coronary artery segments was measured by quantitative angiography. RESULTS: Infusion of L-arginine caused significant dilatation of both proximal (4.87 (0.96)%, p < 0.01 v normal saline) and distal (6.33 (1.38)%, p < 0.01 v normal saline) coronary segments. Co-infusion of vitamin C and L-arginine dilated proximal coronary segments by 8.68 (1.40)% (p < 0.01 v normal saline, p < 0.01 v L-arginine) and distal segments by 13.07 (2.15)% (p < 0.01 v normal saline, p < 0.01 v L-arginine). Intravenous infusion of vitamin C caused a borderline increase in proximal and distal coronary segment diameters (1.93 (0.76)% and 2.09 (1.28)%, respectively, not significant). CONCLUSIONS: L-arginine dependent coronary segment vasodilatation was augmented by the antioxidant vitamin C in patients with coronary artery disease. Thus, vitamin C may have beneficial effects on nitric oxide bioavailability induced by L-arginine.

Effects of atorvastatin on reactive hyperaemia and the thrombosis-fibrinolysis system in patients with heart failure.

OBJECTIVE: To investigate the effects of short term atorvastatin treatment on forearm vasodilatory response to reactive hyperaemia (RH%) and on components of the thrombosis-fibrinolysis system (antithrombin III, proteins and S, factors V and VII, von Willebrand factor, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI-1)) in patients with heart failure. PATIENTS AND METHODS: 35 patients with heart failure were enrolled in this study; 17 patients received atorvastatin 10 mg/day and 18 patients received no statin for four weeks. Forearm blood flow (FBF) was measured by venous occlusion strain gauge plethysmography. RH% and forearm vasodilatory response to nitrate were defined as the percentage change of FBF from rest to the maximum flow during reactive hyperaemia and after nitrate administration, respectively. Plasma concentrations of antithrombin III, protein C, protein S, factor V, factor VII, von Willebrand factor, tPA, and PAI-1 were determined before and after treatment. RESULTS: Maximum hyperaemic FBF remained unchanged in both groups. Baseline FBF was slightly but not significantly decreased in the atorvastatin treated group. RH% was significantly increased only in the atorvastatin treated group, from mean (SD) 42.44 (18.9)% to 83.7 (36.1)% (p < 0.01). Plasma concentrations of antithrombin III (from mean (SD) 81.7 (11.37)% to 73.5 (13.8)%), protein C (from mean (SD) 88.3 (26.9)% to 63.9 (25.0)%), factor V (from mean (SD) 126.2 (33.4)% to 94.9 (29.8)%), tPA (from median (25th-75th percentile) 11.68 (8.60-20.95) ng/ml to 10.30 (8.65-15.12) ng/ml), and PAI-1 (from median (25th-75th percentile) 3.10 (2.15-4.40) IU/l to 1.90 (0.75-3.0) IU/l) were significantly decreased in the atorvastatin treated group (p < 0.05) but not in the control group. Plasma concentrations of von Willebrand factor, factor VII, and protein S remained unaffected in both groups. CONCLUSION: Atorvastatin did not change the maximum hyperaemic flow, although it decreased plasma concentrations of antithrombin III, protein C, factor V, tPA, and PAI-1 in patients with heart failure. Therefore, short term treatment with atorvastatin may affect the expression of both endothelium and liver derived components of the thrombosis-fibrinolysis system in patients with heart failure.

Vascular endothelium and inflammatory process, in patients with combined Type 2 diabetes mellitus and coronary atherosclerosis: the effects of vitamin C.

AIMS: Type 2 diabetes mellitus (DM) and coronary artery disease (CAD) are both associated with endothelial dysfunction and elevated oxidative and inflammatory state. We examined the effect of vitamin C on endothelial function and levels of soluble vascular cell adhesion molecule (sVCAM-1), interleukin-6 (IL-6) and tumour necrosis factor (TNF-alpha), in DM patients with or without CAD and in non-diabetic subjects. METHODS: Thirty-seven patients with DM + CAD, 17 patients with DM without CAD and 21 non-diabetic subjects were divided into groups receiving vitamin C 2 g/day or no anti-oxidant for 4 weeks. Forearm blood flow was determined using venous occlusion gauge-strain plethysmography. Forearm vasodilatory response to reactive hyperemia was considered as index of endothelium-dependent dilation. RESULTS: Baseline levels of IL-6 and TNF-alpha were significantly higher in patients with DM + CAD compared with patients with DM (P < 0.01) or non-diabetic subjects (P < 0.01). IL-6 and TNF-alpha levels were also higher in DM compared with non-diabetic subjects (P < 0.05). sVCAM-1 levels were lower in non-diabetic controls compared with DM + CAD (P < 0.05) or DM (P < 0.05). Reactive hyperaemia was higher in non-diabetic controls compared with DM + CAD (P < 0.001) or DM (P < 0.001). Vitamin C significantly increased reactive hyperaemia only in the DM + CAD group, while it had no effect on serum levels of sVCAM-1, TNF-alpha and IL-6 in any of the groups. CONCLUSIONS: Type 2 diabetes mellitus is associated with impaired endothelial function and increased levels of TNF-alpha, IL-6 and sVCAM-1, especially in patients with DM and CAD. Vitamin C significantly increased forearm vasodilatory response to reactive hyperaemia only in patients with combined DM and CAD.

Effects of L-arginine on flow mediated dilatation induced by atrial pacing in diseased epicardial coronary arteries.

OBJECTIVE: To examine the effects of L-arginine on basal coronary tone and flow mediated dilatation induced by atrial pacing in patients with coronary artery disease and stable angina. DESIGN: Atrial pacing was performed during intracoronary infusions of normal saline and L-arginine (150 micromol/min) in 8 patients with coronary artery disease and stable angina. The luminal diameter of epicardial coronary arteries was assessed by quantitative angiography. RESULTS: L-arginine administration significantly increased the diameter of all the coronary segments and stenoses. During atrial pacing with saline infusion, luminal diameter of the proximal, distal, and stenosis reference segments increased significantly (p < 0.01 versus saline) but stenosis diameter did not change. L-arginine administration did not change the magnitude (NS) of atrial pacing induced dilatation in proximal and distal segments and in coronary stenoses and their reference segments. CONCLUSIONS: Non-stenotic segments of diseased coronary arteries dilate in response to atrial pacing but stenoses do not. L-arginine dilates coronary segments and stenoses but does not increase the magnitude of the response to atrial pacing in proximal and distal segments and in coronary stenoses and their reference segments. These findings provide evidence that the shear stress responsive mechanism is absent at stenoses but present in non-stenotic segments of diseased coronary arteries. They also indicate a relative deficiency of L-arginine, except in the shear response mechanism.

Chemokines in patients with ischaemic heart disease and the effect of coronary angioplasty.

Percutaneous coronary transluminal angioplasty (PTCA) may release inflammatory mediators such as chemokines. Monocyte chemoattractant protein-1 (MCP-1) and eotaxin (EOX) are monocyte- and eosinophil-specific chemokines involved in the inflammation and pathogenesis of coronary atherosclerosis. A total of 28 patients undergoing elective PTCA, 20 coronary artery disease (CAD) patients undergoing coronary angiography and 28 healthy controls were studied. In PTCA patients before the procedure, MCP-1 plasma levels (441+/-64 pg/ml) were similar to those of CAD patients (430+/-24 pg/ml), and significantly higher compared with controls (145+/-17 pg/ml, P<0.01). MCP-1 rose significantly after 3 and 6 months following PTCA (696+/-89 and 876+/-86 pg/ml, respectively, P<0.01 vs. before PTCA). EOX plasma levels (155+/-14 pg/ml) were similar to those of CAD patients (157+/-14 pg/ml), but significantly higher compared with controls (83.2+/-10 pg/ml, P<0.05). EOX rose significantly 24 h (273+/-41 pg/ml, P<0.05) but not 3 months after PTCA (160+/-20 and 158+/-19 pg/ml, respectively). These findings indicate that chemokine-induced monocyte- and eosinophil-specific chemoattraction is stimulated in patients with coronary artery disease. MCP-1 levels remain significantly elevated for at least 6 months following elective PTCA, suggesting an inflammatory stimulation.

Vasomotor effects of L- and D-arginine in stenotic atheromatous coronary plaque.

OBJECTIVE: To examine the effects of exogenous L- and D-arginine on coronary stenosis vasomotion in relation to stenosis morphology. DESIGN: Intracoronary infusions of normal saline, L- and D-arginine (50 and 150 micromol/min), and glyceryl trinitrate (250 microg bolus) were given in 24 patients with coronary artery disease and stable angina. Coronary stenoses were classified as smooth or complex (irregular borders). The diameter of the coronary stenoses and their adjacent reference segments was measured by computed quantitative angiography. RESULTS: During L-arginine infusion a larger proportion of complex stenoses than smooth stenoses dilated by >/= 10% (p < 0.01), and the magnitude of dilatation was greater at the site of complex stenoses (p < 0.05). Irrespective of the type of morphology there was a positive correlation (p < 0.01) between the severity of stenoses and the magnitude of vasodilatation to L-arginine. The response to glyceryl trinitrate was similar in the two groups. No significant change was found in either group in response to D-arginine. CONCLUSIONS: In patients with coronary artery disease, coronary stenoses-particularly those of complex morphology-dilate in response to the administration of L-arginine but not D-arginine. This finding is consistent with partial deficiency of the substrate for nitric oxide synthesis, L-arginine, at the site of complex stenoses.

Therapeutic modification of the L-arginine-eNOS pathway in cardiovascular diseases.

L-Arginine is the substrate for nitric oxide production. Endothelium dysfunction could be attributed to L-arginine deficiency or the presence of L-arginine endogenous inhibitors. This hypothesis leads to the assumption that provision of L-arginine could be the key for endothelial function improvement. Many studies have proven that L-arginine has a beneficial effect on endothelium dependent vasoreactivity, as well as on the interaction between vascular wall, platelets and leucocytes. Therefore, individuals with risk factors for atherosclerosis and patients with coronary artery disease or heart failure, could benefit from therapy with L-arginine.

The impact of risk factors for atherosclerosis on the vasomotor effects of inhibition of nitric oxide synthesis in patients with normal angiograms.

We assessed the impact of systematic risk factors on the vasomotor effects of inhibition of nitric oxide synthesis. N(G)-monomethyl-L-arginine (LNMMA) was infused intracoronarily at 4, 8 and 16 micromol/min followed by intracoronary bolus administration of 250 microg nitroglycerin. Computerized angiography was used to assess the changes in the diameter of coronary segments. During the LNMMA infusions there was no significant difference in LNMMA response between smokers and non-smokers (-5.5+/-0.8 and -6.6+/-0.6%, respectively) or between hypertensives and normotensives (-6.4+/-1.1 and -6.1+/-0.6%, respectively), but the response was less in hypercholesterolaemic patients (-4.5+/-0.7 vs. -8.0+/-0.6%, p<0.05). Thus, the reduced nitric oxide activity is related to hypercholesterolaemia but not to smoking and hypertension.

L-Arginine in coronary atherosclerosis.

Nitric oxide is formed from the N-guanido terminal of the amino acid L-arginine and from molecular oxygen by nitric oxide synthase enzymes. L-arginine administration improves the coronary blood flow response to acetylcholine in patients with normal coronary arteries and hypercholesterolemia, reverses the defective endothelium-dependent vasodilation associated with an elevated plasma low-density lipoprotein level or hypercholesterolemia, dilates coronary epicardial arteries and stenoses, enhances nitric oxide generation, and inhibits lesion formation after balloon angioplasty. Stimulation of endogenous nitric oxide production could inhibit atherogenesis, and therefore may be of benefit in patients with risk factors for atherosclerosis.

Complex stenosis morphology and vasomotor responses to inhibition of nitric oxide synthesis.

OBJECTIVE: To assess the relation between coronary vasomotor effects of N(G)-monomethyl-L-arginine (LNMMA) administration and coronary stenosis morphology, length, and severity in patients with stable angina. DESIGN: In 28 patients (24 male, four female) with coronary artery disease and chronic stable angina, intracoronary normal saline and 4 micromol/min LNMMA were infused for four minutes each, followed by an intracoronary bolus of 250 microg glyceryl trinitrate. Coronary stenoses were classified as concentric (smooth), eccentric (smooth), or complicated (irregular). The diameters of these stenoses and their adjacent reference proximal segments were measured by quantitative angiography. RESULTS: During LNMMA infusion a significantly larger proportion of complicated stenoses than concentric and eccentric stenoses constricted by >/= 5% (p < 0.01) and the magnitude of vasoconstriction was greater in complicated than in concentric and eccentric stenoses (p < 0.05). For complicated stenoses the magnitude of constriction (in mm) with reference to normal saline was greater than that of the concentric and eccentric stenoses (p < 0.05), whereas concentric and eccentric stenoses constricted similarly. Irrespective of the type of morphology, there was a correlation (p < 0.05) between both the severity and the length of stenoses and the magnitude of vasoconstriction to LNMMA. A similar proportion of concentric, eccentric, and complicated stenoses showed >/= 5% increase in diameter with glyceryl trinitrate, and the magnitude of the response was similar in the three groups. CONCLUSIONS: In patients with coronary artery disease, the response to LNMMA is greater when stenosis morphology is complex, indicating greater nitric oxide activity. This provides further evidence that plaques with complex morphology are in an active state.

The role of carotid atherosclerosis in the distinction between ischaemic and non-ischaemic cardiomyopathy.

AIM: Sometimes ischaemic cardiomyopathy is a result of severe coronary artery disease of an occult course, without typical symptoms or evidence of myocardial infarction. This form of presentation is usually indistinguishable from non-ischaemic dilated cardiomyopathy. Carotid bifurcation atherosclerosis and coronary artery disease have been shown to be strongly associated. We prospectively examined the value of extracranial carotid atherosclerosis in the distinction between ischaemic and non-ischaemic aetiology in patients with clinically unexplained cardiomyopathy. METHODS AND RESULTS: Seventy-eight patients with undetermined dilatation and diffuse impairment of the left ventricular contraction were studied within 28 months. They underwent carotid scan and coronary arteriography. Carotid atherosclerosis was found to be very common in ischaemic and rare in non-ischaemic cardiomyopathy. The presence of at least one abnormal carotid finding (intima-media thickness >1 mm, plaques, severe carotid stenosis) was 96% sensitive and 89% specific for ischaemic cardiomyopathy. CONCLUSION: Carotid scanning may be a useful screening and decision making tool in patients with cardiomyopathy of indecisive cause. Patients with carotid atherosclerosis are likely to suffer from severe coronary artery disease. Coronary angiography and subsequent myocardial viability studies, when indicated, could be considered early during their evaluation. In contrast, a negative carotid scan predicts non-ischaemic cardiomyopathy.

Serum cholesterol level, cigarette smoking, and vasomotor responses to L-arginine in narrowed epicardial coronary arteries.

We examined the impact of serum cholesterol and cigarette smoking on the coronary vasomotor effects of L-arginine in patients with atherosclerotic coronary artery disease. The dilation of proximal and distal segments in response to low-dose L-arginine was greater in patients with a serum cholesterol level < or =200 mg/dl than in patients with a level >200 mg/dl, whereas the response was the same in smokers and nonsmokers.

Inhibition of nitric oxide synthesis in human epicardial coronary arteries and stenoses in relation to serum lipid level.

Administration of N(G)-monomethyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase, causes a reduction in epicardial coronary artery and stenosis diameter in patients with coronary artery disease, indicating that these diseased vessels produce nitric oxide. Elevations of low density lipoprotein cholesterol impair human endothelium-dependent relaxation. The relationship between serum lipid level and nitric oxide production by normal and atheromatous human epicardial coronary arteries in vivo is unknown. The effects of an intracoronary infusion of LNMMA (8 and 16 micromol/min) followed by intracoronary administration of 250 mcg nitroglycerin on non-stenotic proximal and distal coronary segments and coronary stenoses were studied in 11 patients with coronary artery disease and in 19 patients with 'normal arteriograms'. Coronary luminal diameter was measured by computerized quantitative angiography. In patients with cholesterol level> or = 220 mg/dl, no significant response to LNMMA was observed in the proximal segments in either those with 'normal angiograms' or those with coronary disease. In patients with cholesterol <220 mg/dl significant constriction (P<0.01) was observed in the proximal segments of patients with 'normal coronary angiograms' at both 8 and 16 micromol doses, but occurred only at the 16 micromol/min dose (P<0.01) in those with coronary disease. In conclusion the difference in vasomotor response to LNMMA in relation to cholesterol level is localised to the proximal coronary segments, and the response does not correlate with cholesterol or triglyceride level. This is therefore more likely to be an indirect effect of elevated cholesterol, e.g. undetected atheroma, than a direct effect on nitric oxide synthesis.

Evidence of partially preserved endothelial dilator function in diseased coronary arteries.

OBJECTIVE: To examine the effects of substance P (endothelium dependent vasodilator) and glyceryl trinitrate (endothelium independent vasodilator) on epicardial coronary arteries in patients with normal coronary angiograms and patients with coronary artery disease. DESIGN: Intracoronary infusions of normal saline, the receptor mediated nitric oxide stimulant substance P (5.6 and 27.8 pmol/min each for five minutes), and glyceryl trinitrate (250 microg bolus) were given in 24 patients with coronary artery disease and stable angina, and in nine patients with normal angiograms. The diameter of proximal and distal coronary segments was measured by computerised quantitative angiography RESULTS: Proximal segments of patients with coronary artery disease dilated less than those of patients with normal angiograms in response to 27.8 pmol/min substance P (mean (SEM): 7.9 (1.3)% v 15 (2.3)% respectively, p < 0. 01). The proximal segments of diseased arteries also dilated less than those of "normal" arteries in response to glyceryl trinitrate (10.2 (1.6)% v 18.4 (2.9)%, respectively, p < 0.01). The responses of distal segments to substance P and glyceryl trinitrate were similar in the two patient groups. There were correlations (all p < 0.001) between the coronary diameter after substance P and after glyceryl trinitrate in normal proximal segments (r = 0.94) and normal distal segments (r = 0.64), in diseased proximal segments (r = 0.95) and diseased distal segments (r = 0.89), and for coronary stenoses (r = 0.93). CONCLUSIONS: Proximal segments of patients with coronary disease dilated less than the proximal segments of "normal" patients in response to substance P and glyceryl trinitrate. The response to substance P is substantial and closely correlated with the response to glyceryl trinitrate in both "normal" patients and those with coronary disease. This suggests that although the proximal segments of diseased coronary arteries have a reduced capacity to dilate in response to direct stimulation of smooth muscle cell relaxation, they retain much of their endothelium dependent vasodilator function.

Effects of L- and D-arginine on the basal tone of human diseased coronary arteries and their responses to substance P.

OBJECTIVE: To assess the effects of substance P administration alone and in combination with L- and D-arginine in patients with normal angiograms and in patients with coronary artery disease. DESIGN: Intracoronary infusions of (a) normal saline, (b) the receptor mediated nitric oxide stimulant substance P (5.6 and 27.8 pmol/min) before and after L- or D-arginine (50 and 150 micromol/min), and (c) glyceryl trinitrate (250 microg bolus) were given to 17 patients with coronary artery disease and stable angina, and to six patients with normal angiograms. The diameter of angiographically normal proximal and distal segments and coronary stenoses were measured by computerised quantitative angiography. RESULTS: L-arginine administration was associated with significant dilatation of stenoses (p < 0.01) of proximal segments of both "normal" (p < 0.05) and diseased (p < 0.01) arteries, and of distal segments of diseased arteries (p < 0.01). No significant changes were associated with D-arginine administration. Dose dependent dilatation of all segments including stenoses, was observed with substance P both before and after L-arginine infusion (p < 0.01). The magnitude of dilatation of stenoses and all segments of both "normal" and diseased coronaries was greater after L-arginine (p < 0.05) but not D-arginine and substance P infusion, than it was after saline and substance P infusion. Administration of D- or L-arginine did not change the magnitude of substance P induced dilatation. CONCLUSIONS: Diseased and "normal" coronary arteries dilated in response to substance P and L-arginine but were unaffected by D-arginine infusion. The magnitude of the response to substance P was not increased by L-arginine administration, indicating that it is not critically dependent on the availability of substrate for nitric oxide synthase.