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The benefits of antithrombotic, and in particular antiplatelet, therapy in patients with atherosclerotic cardiovascular disease are well established, the accumulated evidence now stretching back well over three decades. What has been more difficult to pin down is the optimal intensity as well as duration of such therapy in different clinical situations, since the undoubted benefits (in terms of prevention of thrombotic complications) have to be balanced against the harms (and in particular bleeding complications) that come with it. In primary prevention, the weight of evidence suggests that there is little to be gained by antiplatelet therapy, since the relatively small reduction in thrombotic risk is matched (or even exceeded) by the bleeding risk; whereas by contrast, following acute coronary syndrome, a situation where re-thrombotic risk is high, the benefit gained by one year of dual antiplatelet therapy (DAPT) with aspirin combined with a potent P2Y12 inhibitor greatly outweighs the risk from bleeding (although, in patients considered at especially high bleeding risk, there is reason to reduce the intensity and/or length of DAPT). These considerations are reflected in the most recent international guidelines, including from the European Society of Cardiology [ [1] ] and the American College of Cardiology / American Heart Association [ [2] ].

Background Despite the availability of aggressive lipid-lowering strategies, many patients remain at risk of cardiovascular events. C-reactive protein is a marker of inflammation elevated in patients at high risk of cardiovascular events. C-reactive protein has demonstrated value as a predictor of cardiovascular risk; however, it is unclear whether targeting C-reactive protein levels improves outcomes. This systematic review aimed to characterise the relationship between C-reactive protein and cardiovascular outcomes and to assess whether the magnitude of C-reactive protein reduction correlates to the extent of cardiovascular risk reduction. Methods A systematic review was conducted to identify randomised controlled trials that measured C-reactive protein before and after administration of therapies for cardiovascular disease and measured incidence of cardiovascular events. A meta-analysis of placebo-controlled studies assessed the relationship between extent of C-reactive protein reduction and cardiovascular risk reduction. Placebo-controlled studies where low-density lipoprotein and triglyceride data were available were also included in a meta-regression to assess the influence of these established risk factors on the efficacy of treatment when compared to C-reactive protein. Results Fifteen studies met the criteria for inclusion in this review, of which six were active comparator studies and nine were placebo controlled. Six placebo-controlled studies had data available for meta-regression. Eight studies demonstrated a reduction in events that could be explained by changes in lipid levels, whereas the results of five studies suggested that the association between C-reactive protein reduction and event rates cannot be explained by changes in lipid levels alone. No correlation was found between magnitude of C-reactive protein reduction and cardiovascular risk reduction. A strong correlation was found between C-reactive protein and low-density lipoprotein reduction (adjusted r2 = 0.8). Conclusions Targeting C-reactive protein does not offer additional benefit over targeting low-density lipoprotein across the general population in terms of cardiovascular risk reduction. However, there is value in targeting C-reactive protein in patients at high residual inflammatory risk despite non-elevated lipid levels or use of lipid-lowering therapy.

Aims The aim was to perform a systematic review of the efficacy of traditional Chinese medicines (TCM) in cardiovascular disease. Methods Electronic databases were searched up to 11 November 2015 for all randomized-controlled trials evaluating the effect of TCM in hypertension, ischaemic stroke, heart failure, coronary heart disease and type 2 diabetes mellitus. Pooled odds ratios (ORs) were calculated using a fixed-effects model. Results Four hypertension studies were eligible for statistical analysis and included 133 patients receiving TCM and 130 control patients. There were significant reductions in systolic blood pressure in patients receiving TCM, comparable to results achieved with pharmaceutical medicines. An OR of 3.781 (95% confidence interval 2.392, 5.977; P = 0.000) was observed for the anti-hypertensive effect of TCM. Significant heterogeneity was present (P = 0.011), with a tendency towards publication bias that did not reach significance (P = 0.05275). Outcome measures for other cardiovascular diseases were inconsistent. Conclusions Certain TCM compounds appear to have significant anti-hypertensive effects, and although some are associated in some studies with improved outcomes in coronary heart disease, heart failure and type 2 diabetes mellitus, the data are inconsistent and will require large-scale randomized-controlled trials to allow full evaluation of any potential therapeutic benefit in these areas.

Aims Antiplatelet therapy reduces the incidence of ischaemic stroke. Platelet-mediated thrombosis contributes variably to the major subtypes of stroke as defined by the TOAST criteria: large artery atherosclerosis (LAA), cardioembolic (CE) and small vessel occlusion (SVO). The effect of antiplatelet therapy on the incidence of each subtype is unknown and is the subject of this meta-analysis. Methods Electronic databases were searched for articles comparing the effect of antiplatelet therapy on the incidence of stroke according to aetiological subtype. Studies containing subjects prescribed anticoagulant therapy or solely investigating subjects with atrial fibrillation were excluded. Pooled odds ratios (ORs) were calculated using a fixed effects model. Results Nine studies were included (n = 5739). In patients who had an ischaemic stroke, pre-event antiplatelet therapy was associated with significantly decreased incidence of LAA (OR 0.88, 95% CI 0.79, 0.99; P = 0.026), increased incidence of CE (OR 1.23, 95% CI 1.08, 1.41; P = 0.002) and no effect on SVO (OR 0.99, 95% CI 0.88, 1.11; P = 0.806). Concordant non-significant trends were observed in primary prevention populations (n = 751): LAA (OR 0.81, 95% CI 0.57, 1.15; P = 0.240), CE (OR 1.29, 95% CI 0.89, 1.87; P = 0.179) and SVO (OR 0.99, 95% CI 0.73, 1.36; P = 0.970). Subgroup analysis of aspirin monotherapy (n = 3786) demonstrated a significant reduction in LAA (OR 0.87, 95% CI 0.76, 1.00; P = 0.046), but non-significant effects on the incidence of CE (OR 1.17, 95% CI 0.99, 1.39; P = 0.068) and SVO (OR 1.04, 95% CI 0.91, 1.20; P = 0.570). Probability of publication bias was low (P > 0.05). Conclusions Antiplatelet therapy preferentially reduces the incidence of LAA stroke compared with CE and SVO subtypes.

Aim The PlA1/A2 polymorphism of glycoprotein IIIa (GPIIIa) has been associated with both antiplatelet drug resistance and increased cardiovascular events. The aim of this study was to conduct the first meta-analysis investigating the association between carriage of the PlA2 allele and resistance to currently licensed antiplatelet drugs. Methods Electronic databases (MEDLINE and EMBASE) were searched for all articles evaluating genetic polymorphisms of GPIIIa. For studies where antiplatelet resistance was measured using validated techniques, pooled odds ratios (ORs) were calculated using fixed effects and random effects models. Results Sixteen studies were eligible for statistical analysis and included 1650 PlA1 homozygous subjects and 668 carriers of the PlA2 allele. For carriers of the PlA2 allele, OR 0.924 (n = 2318; 95% CI 0.743, 1.151; P = 0.481) was observed for resistance to any antiplatelet drug, OR 0.862 (n = 2085; 95% CI 0.685, 1.086; P = 0.208) for resistance to aspirin and OR 1.429 (n = 233; 95% CI 0.791, 2.582; P = 0.237) for resistance to clopidogrel. In the aspirin cohort, sub-group analysis revealed no statistical association in either healthy subjects or those with cardiovascular disease. PlA2 carriage was marginally associated with aspirin sensitivity using the fixed effects model when identified by the PFA-100 assay (n = 1151; OR 0.743, 95% CI 0.558, 0.989; P = 0.041) but with significant heterogeneity (I2 = 55%; P = 0.002). Significance was lost with analysis using a random effects model. Conclusions The totality of published data does not support an association between carriage of the PlA2 allele and antiplatelet drug resistance. Significant heterogeneity indicates the need for larger studies using validated and standardized assays.

Hypertension is a multifactorial condition which makes the development of treatment approaches difficult. The vast majority of patients are treated with lifestyle measures either alone or in combination with antihypertensive drugs, and this approach is largely successful in controlling blood pressure. However, for a subgroup of patients, control of blood pressure remains resistant to this approach and therefore the development of new strategies is imperative. The sympathetic nervous system has been known to be implicated in hypertension for many decades, and evidence from studies in the past has revealed the benefit of reducing sympathetic nerve activity in the control of blood pressure albeit with severe side effects. Recent technological advances have allowed for specific targeting of the renal sympathetic nerves by catheter ablation. The Symplicity HTN-1 and HTN-2 trials have provided strong evidence for renal denervation giving rise to considerable blood pressure reductions in treatment-resistant hypertensives and, due to the high incidence of hypertension worldwide, this carries the promise of further reducing the global burden of hypertension and its attendant complications. Here we review the evidence for renal denervation in the management of hypertension.

Background This study was to investigate the effect of integrin-linked kinase (ILK) on the transplantation efficiency of stem cell antigen-1-positive cardiac progenitor cells (Sca-1 CPCs) in a mouse myocardial infarction (MI) model. Methods Sca-1 CPCs were isolated from C57/BL6 mice heart tissues and genetically modified with adenovirus vector containing green fluorescent protein (GFP)/ILK or GFP. Cell viability, migration, DNA synthesis, proliferation, and apoptosis were assessed in vitro. Immediately after MI, treated animals received 5×10 GFP-CPC or ILK-CPC transplantation into the peri-infarct myocardium. Cardiac function, exercise ability, cardiac morphology, angiogenesis, cardiomyocyte apoptosis, as well as ILK-related protein expression were measured. Acute and long-term cell survival after cell transplantation was assessed. Results Overexpression of ILK increased the viability, migration, DNA synthesis, proliferation, and survival of Sca-1 CPCs in vitro. Protein expression of phosphorylated Akt and cyclin D1 were up-regulated. In our in vivo experiment, more transplanted cells were found in the peri-infarct myocardium in ILK-CPC group 3 days after cell transplantation, but there was no difference between the two groups 4 weeks later. ILK-CPC group showed reduced infarct size 7 days after cell transplantation. Long-term observation showed improved cardiac function indicated by higher percent fractional shortening and lower left ventricular end systolic diameter/left ventricular end diastolic diameter, better exercise ability, increased angiogenesis, decreased fibrosis and apoptosis, as well as up-regulation of ILK, Cdc42, and Aurora B protein expression in ILK-CPC group 4 weeks after cell transplantation. Conclusions ILK-overexpressed Sca-1 CPCs showed improved therapeutic efficacy in MI.

Hydrogen sulfide (H2S) has been shown to have powerful antioxidative and anti-inflammatory properties that can regulate multiple cardiovascular functions. However, its precise role in diabetes-accelerated atherosclerosis remains unclear. We report here that H2S reduced aortic atherosclerotic plaque formation with reduction in superoxide (O2 -/-) generation and the adhesion molecules in streptozotocin (STZ)-induced LDLr-/- mice but not in LDLr-/-Nrf2-/- mice. In vitro, H2S inhibited foam cell formation, decreased O2 - generation, and increased nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and consequently heme oxygenase 1 (HO-1) expression upregulation in high glucose (HG) plus oxidized LDL (ox-LDL)-treated primary peritoneal macrophages from wild-type but not Nrf2-/- mice. H2S also decreased O2 -/- and adhesion molecule levels and increased Nrf2 nuclear translocation and HO-1 expression, which were suppressed by Nrf2 knockdown in HG/ox-LDL-treated endothelial cells. H2S increased S-sulfhydration of Keap1, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and inhibited O2 -/- generation, which were abrogated after Keap1 mutated at Cys151, but not Cys273, in endothelial cells. Collectively, H2S attenuates diabetes-accelerated atherosclerosis, which may be related to inhibition of oxidative stress via Keap1 sulfhydrylation at Cys151 to activate Nrf2 signaling. This may provide a novel therapeutic target to prevent atherosclerosis in the context of diabetes.

Background Hydrogen sulfide (H 2 S) is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel‐like factor 5 (KLF5) exerts diverse functions in the cardiovascular system. Whether and how H 2 S regulates KLF5 in myocardial hypertrophy is unknown. Methods and Results In our study, hypertrophic myocardial samples in the clinic were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats and neonatal rat cardiomyocytes were studied for functional and signaling responses to GYY4137, an H 2 S‐releasing compound. Expression of cystathionine γ‐lyase, a principal enzyme for H 2 S generation in heart, decreased in human hypertrophic myocardium, whereas KLF5 expression increased. After GYY4137 administration for 4 weeks, myocardial hypertrophy was inhibited in spontaneously hypertensive rats, as demonstrated by improvement in cardiac structural parameters, heart mass, size of cardiac myocytes, and expression of atrial natriuretic peptide. H 2 S diminished expression of KLF5 in myocardium of spontaneously hypertensive rats and in hypertrophic cardiomyocytes. H 2 S also inhibits platelet‐derived growth factor A promoter activity, decreased recruitment of KLF5 to the platelet‐derived growth factor A promoter, and reduced atrial natriuretic peptide expression in angiotensin II–stimulated cardiomyocytes, and these effects are suppressed by KLF5 knockdown. KLF5 promoter activity and KLF5 expression was also reversed by H 2 S. H 2 S increased the S‐sulfhydration on specificity protein 1 in cardiomyocytes. Moreover, H 2 S decreased KLF5 promoter activity; reduced KLF5 mRNA expression; attenuated specificity protein 1 binding activity with KLF5 promoter; and inhibited hypertrophy after specificity protein 1 mutated at Cys659, Cys689, and Cys692 but not Cys664 overexpression. Conclusions These findings suggest that H 2 S regulates KLF5 transcription activity via specificity protein 1 S‐sulfhydration at Cys664 to prevent myocardial hypertrophy.

Objectives. We investigated whether amlodipine could improve angiogenic responses in a diabetic rat model of acute myocardial infarction (AMI) through improving bone marrow endothelial progenitor cell (EPC) mobilization, in the same way as angiotensin converting enzyme inhibitors.Methods. After induction of AMI by coronary artery ligation, diabetic rats were randomly assigned to receive perindopril (2 mgkg−1 day−1), amlodipine (2.5 mgkg−1 day−1), or vehicle by gavage (n=20per group). Circulating EPC counts before ligation and on days 1, 3, 5, 7, 14, and 28 after AMI were measured in each group. Microvessel density, cardiac function, and cardiac remodeling were assessed 4 weeks after treatment. The signaling pathway related to EPC mobilization was also measured.Results. Circulating EPC count in amlodipine- and perindopril-treated rats peaked at day 7, to an obvious higher level than the control group peak which was reached earlier (at day 5). Rats treated with amlodipine showed improved postischemia neovascularization and cardiac function, together with reduced cardiac remodeling, decreased interstitial fibrosis, and cardiomyocyte apoptosis. Amlodipine treatment also increased cardiac SDF-1/CXCR4 expression and gave rise to activation of VEGF/Akt/eNOS signaling in bone marrow.Conclusions. Amlodipine promotes neovascularization by improving EPC mobilization from bone marrow in diabetic rats after AMI, and activation of VEGF/Akt/eNOS signaling may in part contribute to this.

BACKGROUND AND PURPOSE One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O2•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. EXPERIMENTAL APPROACH HUVECs were incubated with ox-LDL with/without pyridoxine, NG-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O2•− was measured using lucigenin chemiluminescence, and O2•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot. KEY RESULTS Ox-LDL significantly increased O2•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O2•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. CONCLUSIONS AND IMPLICATIONS Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.

Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.

Differential effects of beta-adrenoreceptor antagonists (beta-ARB) on central and peripheral blood pressure may relate to change in heart rate and/or vasodilator tone and thus be exaggerated during exercise.To examine acute effects of selective and nonselective beta-ARB on central and peripheral blood pressure, cardiac output and peripheral vascular resistance during exercise.Healthy volunteers (n= 20, 18 men, 19-54 years) received propranolol 80 mg, bisoprolol 20 mg, and placebo 1 h before bicycle ergometry (50, 75 and 100 W each for 3 min) in a randomized, cross-over study. Cardiac output was determined by pulmonary uptake of soluble and inert gas tracers (InnoCor, Innovision). Central systolic blood pressure (SBP) was determined from the late systolic shoulder of the digital artery pressure waveform (Finometer, Finopres).At rest, both beta-ARB reduced brachial but not central SBP (compared with placebo). During exercise, beta-ARB reduced brachial SBP (reductions of 19.9 +/- 4.3 mmHg and 23.2 +/- 2.7 mmHg for propranolol and bisoprolol, respectively, at 100 W, each P0.0001) but not central SBP. The difference between peripheral and central SBP was reduced, relative to that during placebo, by 21.5 mmHg (95% confidence interval 8.8, 34.1) and 26.4 mmHg (18.1, 34.8) for propranolol and bisoprolol, respectively, at 100 W (each P0.01). There was no significant effect of beta-ARB on diastolic blood pressure or peripheral vascular resistance.Despite reducing brachial blood pressure, acute beta-adrenoreceptor blockade in man at rest and during exercise does not reduce central blood pressure.

Essential hypertension is associated with increased risk of arterial thrombotic disease. Among other factors, enhanced platelet activity contributes significantly to this phenomenon. An increased level of circulating monocyte-platelet aggregates (MPAs) represents one of the most robust markers of platelet activation; furthermore, these aggregates are also believed to contribute to the pathophysiology of atherothrombotic disease. Putative mechanisms that contribute to platelet activation in essential hypertension include endothelial dysfunction, neurohumoral (sympathetic and renin-angiotensin systems) overactivity, decreased platelet nitric oxide (NO) biosynthesis, and platelet degranulation secondary to increased shear. Current recommendations are that hypertensive patients receive aspirin therapy only if their calculated cardiovascular risk is high and their blood pressure (BP) is adequately controlled. By contrast, the use of antiplatelet treatment in low-risk hypertensive patients is not established and merits further investigation. Moreover, the place of alternative antiplatelet agents other than aspirin, such as clopidogrel, is unclear at present. Some experimental evidence suggests that clopidogrel may confer an additive protective effect over and above aspirin in hypertensive patients, by virtue of effects on the evolution of the atherosclerotic process. This now needs to be investigated in long-term clinical outcome studies.

Patients with heart failure and underlying ischemic heart disease (IHD) exhibit both endothelial dysfunction and increased arterial stiffness. We investigated whether this is also the case in heart failure of nonischemic etiology.Eleven patients with heart failure and IHD, 12 patients with heart failure from angiographically verified idiopathic nonischemic dilated cardiomyopathy (DCM), and 16 healthy subjects of similar age and sex were compared. Endothelium-dependent and independent function were evaluated by ultrasonic measurement of flow-mediated dilatation (FMD) and glyceryl trinitrate (GTN)-induced dilatation of the brachial artery respectively. Vascular compliance was assessed by carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx). Heart failure severity was similar in IHD and DCM patients. FMD was impaired in the subjects with IHD as compared with control subjects (4.8 +/- 0.3 vs. 8.0 +/- 3.6 %, P.01), but not in those with DCM. GTN-induced vasodilatation was not different in patients and controls. PWV was also increased in IHD patients compared with controls (12.1 +/- 3.6 vs. 8.0 +/- 1.6 m/s, P.01), but not in DCM patients. AIx was similar in patients and controls.Heart failure of nonischemic etiology is not associated with abnormalities of endothelium-mediated dilatation or of arterial compliance. The findings of our study now need to be confirmed in larger studies.

1. Circulating bone marrow (BM)-derived endothelial progenitor cells (EPCs) play an important role in neovascularization. In the present study, we investigated the mechanisms underlying the reduction in circulating EPCs in a mouse model of diabetes induced by streptozotocin. 2. Compared with non-diabetic controls, diabetic mice had reduced circulating EPCs (0.59 +/- 0.11 vs 0.94 +/- 0.21%, respectively; P < 0.01) and increased plasma endothelial microparticles (18 642 +/- 6809 vs 5692 +/- 1862/mL, respectively; P < 0.01). In a mouse bone marrow (BM) transplantation model, increased adhesion of transplanted BM cells to aortas of diabetic mice was observed compared with control (900 +/- 194 vs 431 +/- 109 cells/mm(2), respectively; P < 0.01). 3. Following hindlimb ischaemia, diabetic mice exhibited suppressed EPC mobilization, a reduction in the expected increase in capillary density and suppressed restoration of transcutaneous oxygen pressure in the ischaemic tissue. Diabetic mice also showed impaired ischaemia-induced upregulation of vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1 alpha and interleukin-1 beta, an exaggerated increase in matrix metalloproteinase (MMP)-2 and -9 and a suppressed increase in tissue inhibitor of matrix metalloproteinase (TIMP)-1. On multivariate analysis, VEGF expression was the only independent factor related to circulating EPC count. 4. In conclusion, the data indicate that the decrease in basal circulating EPCs in diabetes may be attributable, in part, to consumptive loss of EPCs due to increased endothelial damage. Impairment of ischaemia-induced EPC mobilization in the diabetic mouse model is associated with altered HIF-1 alpha/VEGF and MMP/TIMP regulation and represents a novel mechanism underlying defective postischaemic neovascularization in diabetes.

Background— Left ventricular (LV) remodeling is associated with the development of heart failure after myocardial infarction. Here we investigated whether integrin-linked kinase (ILK) may regulate LV remodeling and function after myocardial infarction. Methods and Results— Adenoviral vector expressing ILK (n=25) or empty adeno-null (n=25) was injected into rat peri-infarct myocardium after left anterior descending coronary artery ligation. ILK expression was confirmed by Western blotting and immunofluorescence. Echocardiographic and hemodynamic analyses demonstrated relatively preserved cardiac function in adeno-ILK animals. ILK treatment was associated with reduced infarct scar size, increased scar thinning ratio, and preserved LV diameter, wall thickness, cardiomyocyte size, and myofilament density. Enhanced angiogenesis and reduced fibrosis were observed in the adeno-ILK group, along with reduced apoptosis as demonstrated by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling analysis. Moreover, increased cardiomyocyte proliferation was found in adeno-ILK animals, as measured by proliferating cell nuclear antigen, Ki-67, and phosphohistone-H3 staining. At long-term follow-up, most indices of cardiac function and hemodynamics showed no difference between adeno-ILK and control animals by 9 weeks, although LV end-systolic diameter and infarct scar size were reduced in the adeno-ILK group at this time point. Additionally, ILK overexpression was found to exert a rescue effect on remodeling when administered in a delayed fashion 1 week after coronary artery ligation. Conclusions— ILK gene therapy improves cardiac remodeling and function in rats after myocardial infarction and is associated with increased angiogenesis, reduced apoptosis, and increased cardiomyocyte proliferation. This may represent a new approach to the treatment of postinfarct remodeling and subsequent heart failure.

Aims The aim of this study was to determine the effect of blood pressure (BP) on platelet nitric oxide (NO) signalling and on formation of circulating monocyte-platelet aggregates (MPA), as well as the role of platelet NO in modulating MPA in hypertension. Methods and results We first examined platelet NO signalling in 23 untreated hypertensive (UH) and 23 normotensive (NT) subjects. Platelets from hypertensives exhibited reduced NO synthase activation by albuterol or collagen, as well as suppressed basal and stimulated NO-attributable cyclic guanosine-3′,5′-monophosphate, compared with NT. In a second study, comprising 106 subjects with a wide BP range, circulating MPA showed a strong positive correlation with BP. On multiple regression analysis, using a model incorporating systolic BP (SBP), diastolic BP, age, lipids, gender, and smoking status, the only independent predictor of MPA was SBP. Nitric oxide synthase inhibition with N G-monomethyl-l-arginine increased MPA in NT but not in hypertensives, whereas the NO donor spermine NONOate (SNO) decreased MPA in NT but not in hypertensives. Platelet P-selectin expression was higher in hypertensives than in NT, and its expression was suppressed by SNO in NT only. Conclusion Platelet NO production and responsiveness are suppressed with raised BP, and this may contribute to the increase in platelet P-selectin and hence in circulating MPA in hypertension.

Heart failure is a major cause of cardiovascular morbidity and mortality and its incidence is on the increase. The pathophysiology of heart failure is multi-factorial but recent studies suggest that aldosterone plays an important and independent role in its progression. Emerging evidence now suggests that aldosterone exerts renal-independent effects. It binds to its mineralocorticoid receptor to produce direct effects on the myocardium and vasculature, leading to damaging processes such as hypertrophy, necrosis, fibrosis and endothelial dysfunction, factors known to contribute to the pathophysiology of heart failure. Mineralocorticoid receptor antagonists have thus emerged as a new paradigm for the treatment of heart failure. The benefits of these agents on both morbidity and mortality when used in patients with chronic symptomatic heart failure have been demonstrated by recent trials.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • A genetic basis for aspirin resistance has been postulated to exist. • The individual studies published have been too small to allow reliable conclusions to be drawn. • In order to clarify what, if any, genetic basis exists for aspirin resistance, we have undertaken, to our knowledge, the largest and most comprehensive systematic review conducted to-date in this field. WHAT THIS STUDY ADDS • There is a significant association between the PlA1/A2 molecular variant and aspirin resistance in healthy subjects, with the effect diminishing in the presence of cardiovascular disease. • However further studies are needed to confirm this. • We argue that investigators now need to agree on a standard technique to measure and define aspirin resistance. AIMS The aim was to perform a systematic review of all candidate gene association studies in aspirin resistance. METHODS Electronic databases were searched up until 1 December 2007 for all studies investigating any candidate gene for aspirin resistance in humans. Aspirin resistance was required to have been measured by a standardized laboratory technique to be included in the analysis. RESULTS Within 31 studies, 50 polymorphisms in 11 genes were investigated in 2834 subjects. The PlA1/A2 polymorphism in the GPIIIa platelet receptor was the most frequently investigated, with 19 studies in 1389 subjects. The PlA1/A2 variant was significantly associated with aspirin resistance when measured in healthy subjects [odds ratio (OR) 2.36, 95% confidence interval (CI) 1.24, 4.49; P = 0.009]. Combining genetic data from all studies (comprising both healthy subjects and those with cardiovascular disease) reduced the observed effect size (OR 1.14, 95% CI 0.84, 1.54; P = 0.40). Moreover, the observed effect of PlA1/A2 genotype varied depending on the methodology used for determining aspirin sensitivity/resistance. No significant association was found with aspirin resistance in four other investigated polymorphisms in the COX-1, GPla, P2Y1 or P2Y12 genes. CONCLUSIONS Our data support a genetic association between the PlA1/A2 molecular variant and aspirin resistance in healthy subjects, with the effect diminishing in the presence of cardiovascular disease. The laboratory methodology used influences the detection of aspirin resistance. However, as heterogeneity was significant and our results are based on a limited number of studies, further studies are required to confirm our findings.

Objective Vascular endothelial nitric oxide (NO) biosynthesis is reported to be decreased in patients with atherosclerosis. The primary aim of the present study was to determine whether whole body NO production is decreased in patients with established coronary atherosclerosis (CA) as compared to healthy control (HC) subjects. As a secondary aim, we wished to ascertain whether whole body NO biosynthesis is inversely related to plasma homocysteine (Hcy) levels. Design Whole body NO production was assessed by measuring the amount of [15N]-nitrate excreted in urine, following intravenous administration of l -[15N]2-arginine. Subjects 19 CA and 13 HC. Results Mean urinary [15N]-nitrate excretion was not different between the CA (113.1 ± 13.9 nmol/mmol creatinine) and HC (129.9 ± 15.4 nmol/mmol creatinine) groups, and was not different in CA subjects taking nitrates as compared to those not taking nitrates. Linear regression analysis revealed a strong inverse correlation between [15N]-nitrate excretion and plasma Hcy concentration (r = 0.475, p = 0.012). In contrast, no relationship was observed between [15N]-nitrate excretion and age, blood pressure (systolic or diastolic), plasma cholesterol (including subfractions), triglycerides or glucose. Conclusions Whole body NO production is inversely related to plasma Hcy, but is not related either to established coronary atherosclerosis or to the presence of other cardiovascular risk factors.

Objective: Ageing is associated with an increase in atherothrombotic disease. Platelet-derived nitric oxide (NO) inhibits platelet activation, but the effect of age on platelet NO signaling is unknown. We investigated platelet NO biosynthesis and responsiveness in older (N45 years old) as compared with younger (b30 years old) healthy human subjects. Methods: Platelet NO synthase (NOS) activity was evaluated by l-[ 3 H]-arginine to l-[ 3 H]-citrulline conversion, and cGMP was determined by radioimmunoassay. Platelet expression of NOS3, phosphoserine-1177-NOS3 and soluble guanylyl cyclase (sGC) were quantified by Western blotting. Circulating monocyte–platelet aggregates (MPA) were measured by flow cytometry. Results: Basal NOS activity was similar in both groups. By contrast, whereas both albuterol and collagen stimulated platelet NOS in younger subjects, stimulation was absent in older subjects. Platelet NOS3 expression was similar in both age groups, but NOS3 serine-1177 phosphorylation was greater in younger subjects. Basal, albuterol- and collagen-stimulated cGMP, as well as sGC expression, were all greater in younger than older subjects, and within the younger group both cGMP (basal and stimulated) and sGC expression were greater in women than in men. Circulating MPA were greater in older subjects and, whilst NOS inhibition increased MPA further in both groups, it did so to a lesser extent in the older age bracket. Conclusions: These data suggest that platelet NO production and responsiveness decrease with age, and this is reflected in increased circulating MPA.

Background and purpose: Diabetes mellitus, especially type 2, is associated with increased arterial thrombosis. Our aims were (i) to characterize and compare platelet aggregation in vivo and in vitro in a type 2 diabetes model; and (ii) to determine whether these results differ from those in a type 1 diabetes model. Experimental approach: Platelet aggregation to ADP in lean or obese Zucker Diabetic Fatty (ZDF) rats and in streptozotocin (STZ)-treated or control Wistar rats was measured in vitro, using Born aggregometry, and in vivo, by 111Indium-labelled pulmonary platelet accumulation. Key results: In vivo, ADP responses were higher in obese (type 2 model) than lean ZDF rats. However, in vitro, ADP aggregation did not differ between platelet-rich plasma from ZDF lean or obese rats; nor was any difference seen in ADP responses when platelets from either lean or obese ZDF rats were suspended in plasma from obese or lean ZDF rats, respectively. In vivo, ADP responses were similar in STZ treated (type 1 model) and control rats whereas, in vitro, isolated platelets from STZ diabetic rats were more responsive to ADP aggregation than controls. Platelets from control or STZ-treated rats suspended in plasma from STZ-treated rats exhibited reduced ADP aggregation, compared to when suspended in plasma from control rats. Conclusions and implications: The platelet aggregation results obtained in vitro do not reflect those in vivo, therefore in vitro aggregation data should be interpreted with caution. Moreover, both in vitro and in vivo, different diabetic models exhibit important differences in platelet responsiveness. British Journal of Pharmacology (2007) 150, 105–111. doi:10.1038/sj.bjp.0706957

Vascular disease is the most important complication of both type 1 and type 2 diabetes, with both micro- and macrovascular disease underlying most of the death and disability observed in diabetic patients. Endothelial dysfunction is a cardinal feature of both types of diabetes, and is believed to be involved in the aetiology and pathophysiology of diabetic vasculopathy. Therefore, measures which improve endothelial dysfunction in diabetes are currently the subject of much interest and research. In this article, we review both drug and non-drug therapies for endothelial dysfunction, along with evidence of their effectiveness in diabetes. We suggest that those therapies for which good evidence of endothelial benefit exists should be more widely used in diabetic patients, and that the maximum benefit will be derived by using multiple such therapies in combination.

Low-density lipoprotein (LDL) inhibits endothelium-dependent vasorelaxation. The aim of this study was to determine whether pyridoxine supplementation improves indices of LDL-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVEC) were incubated with native LDL (nLDL) from healthy subjects, oxidized LDL (oxLDL, formed by nLDL oxidation) or nLDL from type II diabetic patients (dLDL), in the absence or presence of pyridoxine; nitric oxide synthase (NOS) activity, cyclic GMP and expression of NOS isoforms were measured, as well as thiobarbituric acid reactive substances (TBARS) in HUVEC supernatants and amino acid concentrations in HUVEC lysates. All LDL species inhibited total NOS activity, whilst increasing the much smaller Ca2+-independent component of NOS activity, the effects of oxLDL being greatest and those of nLDL smallest; in accordance with these findings, NOS type 3 expression decreased and NOS type 2 expression increased, with a resultant decrease in bioactive nitric oxide (NO), in HUVEC treated with each LDL species, with the same rank order of potency. LDL species also increased TBARS in HUVEC supernatants as well as homocysteine concentrations in HUVEC lysates, nLDL < dLDL < oxLDL. Pyridoxine largely prevented all LDL-induced changes in NOS activity and isoform expression, as well as in TBARS and homocysteine. The findings suggest that pyridoxine prevents LDL-induced dysfunction of endothelial cell NO generation, most likely through its antioxidant effects as well as through its effects on cellular homocysteine metabolism. This has important potential therapeutic implications for cardiovascular disease prevention.

Nitric oxide plays a crucial role in cardiovascular homeostasis, with important vasodilatory, anti-thrombotic and anti-atherogenic properties. Beta-adrenergic receptors (betaARs), present on a wide variety of cardiovascular cells, including vascular endothelial cells, platelets, cardiac myocytes and leukocytes, have long been established as key players in maintaining cardiovascular homeostatic control. During the last few years a wealth of evidence has emerged which directly links stimulation of these cardiovascular betaARs to nitric oxide (NO) generation, suggesting a new and important mechanism of adrenergic control of cardiovascular function. This review explores the cardiovascular cell systems in which this coupling of betaARs and NO occurs, the intracellular signalling and regulatory mechanisms involved and the abnormalities in betaAR-NO oxide coupling found in cardiovascular disease states.

This commentary reviews recent evidence that implicates nitric oxide (NO) as a mediator of β2-adrenoceptor (β2-AR)-initiated vasodilatation. Emphasis is placed on the following: 1) in vivo studies that demonstrate potential physiological importance, 2) mechanistic studies performed in vitro in human umbilical vein endothelial cells (HUVEC), 3) effects of β2 agonists on arterial pulse wave reflection, and 4) therapeutic opportunities offered by the combination of β2 agonist action with selective β1 antagonism. Vascular β2-AR-initiated mechanisms provide a physiologically important control mechanism during exercise. Activation of β2-AR in HUVEC leads to vasodilatation that is partly NO-mediated via activation of protein kinase A (PKA) and of phosphatidylinositol-3 kinase (PI3K)/Akt pathways, leading to serine phosphorylation of the endothelial NO synthase (eNOS). In vivo, β2-AR activation limits the rise in blood pressure during exercise and reduces arterial pulse wave reflection. Nebivolol is a selective β1-AR antagonist with vasodilator actions operating through these pathways, offering novel therapeutic opportunities.

We evaluated, in endothelial cells, the relative effectiveness of l-arginine and l-ascorbate in preventing the decrease in nitric oxide (NO) production in response to native low-density lipoprotein (LDL) from healthy subjects (nLDL), oxidized LDL (oxLDL, formed by nLDL oxidation) or native LDL from type 2 diabetic patients (dLDL). Human umbilical vein endothelial cells (HUVEC) were exposed to nLDL, dLDL or oxLDL (100mgprotein/L), in the absence or presence of l-arginine 10 −4 mol/L and/or l-ascorbate 10 −4 mol/L; NO synthase (NOS) activity and cyclic guanosine-3′,5′-monophosphate (cGMP) were measured by the conversion of l-[3H]arginine to l-[3H]citrulline and by radioimmunoassay, respectively. Both l-arginine and l-ascorbate increased cGMP in HUVEC co-incubated with any LDL species, although to lower levels than found in the absence of LDL. l-Ascorbate did not affect NOS activity, whereas l-arginine increased it, both in the absence and presence of all LDL species. The effects of combined l-arginine and l-ascorbate on NOS activity and cGMP were no greater than those of l-arginine alone. Our results suggest that l-arginine or l-ascorbate can ameliorate, but not normalize, NO production in this situation, and that combining l-arginine with l-ascorbate is unlikely to produce additional benefit as compared with l-arginine alone.

Vasorelaxation to β2-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca2+-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca2+-independent NOS-3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of β2-adrenergic relaxation of rat aorta. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10−7M), the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (5 × 10−7M), Akt inhibitor (10−5M), or vehicle, in the absence or presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10−4M). Rings were then contracted with phenylephrine (10−7M), and concentration–relaxation responses determined to the β2-adrenoceptor agonist albuterol. Rings exhibited a concentration-dependent relaxation to albuterol: pEC50 6.9±0.2, Emax 88.2±4.0%. L-NAME attenuated Emax to 60.2±3.5% (P

1. We investigated the relative effectiveness of L-arginine, L-ascorbate and pyridoxine in preventing the impairment of endothelium-mediated vasorelaxation induced by native low-density lipoprotein (nLDL) from healthy subjects, oxidised LDL (oxLDL, formed by oxidation of nLDL) or nLDL from type II diabetic patients (dLDL). 2. Rabbit aortic rings were exposed to nLDL, dLDL or oxLDL (50-200 mg protein l-1), or corresponding vehicle, following which they were constricted with noradrenaline 10(-6) M; concentration-relaxation curves were determined to acetylcholine (ACh), A23187, or sodium nitroprusside (NP), in the absence or presence of L-arginine (10(-5)-10(-3) M), L-ascorbate (10(-5)-10(-3) M) and pyridoxine (0.5-2.0 mM). 3. nLDL, dLDL and oxLDL all inhibited relaxant responses to ACh and A23187, but not to NP, in a concentration-dependent manner (oxLDL>dLDL>nLDL). 4. In the presence of all LDL preparations, L-arginine, L-ascorbate or pyridoxine each improved ACh and A23187 responses, although none completely normalised endothelium-dependent relaxations. The maximal effect of L-arginine occurred at 10(-4) M. The combination of L-arginine 10(-4) M, L-ascorbate 10(-5) M and pyridoxine 2.0 mM was equally effective as L-arginine 10(-4) M alone. 5. Our results confirm that nLDL, dLDL and oxLDL exert inhibitory effects on endothelium dependent, but not endothelium independent, relaxation of rabbit aorta. ACh and A23187 responses in the presence of any LDL species can be ameliorated by supplementation with L-arginine, L-ascorbate or pyridoxine, either singly or in combination, with no agent or combination proving superior to L-arginine alone. Nevertheless, ACh and A23187 responses are not completely normalised with such supplements, suggesting that there also exists a component of LDL-induced inhibition of endothelium-mediated vasorelaxation that is independent of the nitric oxide system.

Objectives To compare the effects of the sodium-hydrogen exchange blocker, amiloride, with those of the angiotensin-converting enzyme inhibitor, enalapril, on cardiac structure and function and intracardiomyocyte calcium concentration ([Ca2+]i) and pH (pHi), in spontaneously hypertensive rats (SHRs). Methods Experiments were performed in SHRs treated for 4 weeks with amiloride 7.5 mg/kg per day, enalapril 6.0 mg/kg per day or vehicle, and in Sprague-Dawley rats (SDRs). After haemodynamic measurements were taken, the heart was removed and weighed and hydroxyproline (a marker of collagen content) was assayed. In separate rats, ventricular myocytes were isolated, their size determined, and [Ca2+]i and pHi examined using fluo-3 acetoxymethyl ester and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis acetoxymethyl ester fluorescence, respectively. Results Left ventricular end-diastolic pressure was increased, and the maximal rates of increase and of decrease in pressure with time in the left ventricle were decreased in SHRs compared with SDRs. Myocytes were larger and hydroxyproline was increased in the left ventricle, but not in the right ventricle of SHRs compared with SDRs. Amiloride and enalapril decreased systolic blood pressure in SHRs similarly, and improved diastolic function in these rats, enalapril more than amiloride. Both agents decreased left ventricular myocyte size to similar extents; however, whereas enalapril decreased the left ventricular hydroxyproline content, amiloride did not. Left ventricular myocytes from SHRs exhibited greater [Ca2+]i and pHi than those from SDRs; enalapril decreased [Ca2+]i more than amiloride, but amiloride decreased pHi more than enalapril. Conclusions In SHRs, enalapril prevents left ventricular hypertrophy, collagen deposition, diastolic dysfunction, and increases in [Ca2+]i more effectively than does amiloride. In contrast, the latter prevents the increase in pHi more effectively than enalapril, despite similar reductions in blood pressure. These findings suggest that their effects do not depend solely on blood pressure reduction.

We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets.NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration.Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation.Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.

Hydrogen sulfide (H2S) is a gasotransmitter which regulates multiple cardiovascular functions. However, the precise roles of H2S in modulating myocardial fibrosisin vivoand cardiac fibroblast proliferationin vitroremain unclear. We investigated the effect of GYY4137, a slow-releasing H2S donor, on myocardial fibrosis. Spontaneously hypertensive rats (SHR) were administrated with GYY4137 by intraperitoneal injection daily for 4 weeks. GYY4137 decreased systolic blood pressure and inhibited myocardial fibrosis in SHR as evidenced by improved cardiac collagen volume fraction (CVF) in the left ventricle (LV), ratio of perivascular collagen area (PVCA) to lumen area (LA) in perivascular regions, reduced hydroxyproline concentration, collagen I and III mRNA expression, and cross-linked collagen. GYY4137 also inhibited angiotensin II- (Ang II-) induced neonatal rat cardiac fibroblast proliferation, reduced the number of fibroblasts in S phase, decreased collagen I and III mRNA expression and protein synthesis, attenuated oxidative stress, and suppressedα-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) expression as well as Smad2 phosphorylation. These results indicate that GYY4137 improves myocardial fibrosis perhaps by a mechanism involving inhibition of oxidative stress, blockade of the TGF-β1/Smad2 signaling pathway, and decrease inα-SMA expression in cardiac fibroblasts.

We investigated the nitric oxide (NO) dependence of vasorelaxation in response to different calcium channel blockers (CCB), in rabbit femoral artery in vivo. Anaesthetized rabbits underwent femoral artery ligation, and blood from the proximal artery was returned distal to the ligature through a constant infusion pump. The effects of local injection of CCB on perfusion pressure and plasma nitrite+nitrate (NOx, which reflects local NO biosynthesis) concentration in this system were determined. Intra-arterial verapamil, nifedipine or amlodipine 10 μmol kg−1 each reduced perfusion pressure. Pre-treatment with intra-arterial NG-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor) 1 μmol kg−1 did not affect responses to verapamil or nifedipine, but attenuated the reduction in perfusion pressure to amlodipine, from 33.2±2.1% to 22.5±1.6% (P=0.002). Intra-arterial amlodipine – unlike verapamil or nifedipine – increased femoral venous NOx, from 9.1±0.4 μM to 14.1±0.5 μM (P=0.005). The bradykinin B2 receptor antagonist HOE 140, 30 mg kg−1, attenuated the reduction in perfusion pressure and abolished the rise in venous NOx concentration, following intra-arterial amlodipine. Amlodipine potently inhibited serum angiotensin converting-enzyme (ACE) activity in vitro, as effectively as enalapril at similar concentrations. These results suggest that the vasorelaxant effects of nifedipine and verapamil are NO-independent, whereas those of amlodipine are partly NO-dependent, in rabbit femoral artery in vivo. This effect of amlodipine occurs through B2 receptor activation, and may be related to an increase in local bradykinin through inhibition of ACE. British Journal of Pharmacology (2002) 136, 375–382; doi:10.1038/sj.bjp.0704753

What you need to know Antithrombotic medications reduce thromboembolic events by inhibiting platelet aggregation and coagulation. Antiplatelet drugs and oral anticoagulants are examples of antithrombotic medications and are among the most commonly prescribed drugs in both primary and secondary care.1 Clinicians are familiar with their use, however antiplatelets and oral anticoagulants are the drug classes most commonly implicated in adverse drug reactions occurring both in the community and in hospital.23 Increasing numbers of patients have an indication for combination antiplatelet and oral anticoagulant therapy. For example, more than one million people in the UK have atrial fibrillation, of whom approximately one third also have an indication for antiplatelet therapy as secondary prevention.4 Despite the need to understand the balance between benefit and risk, there are limited randomised data investigating antithrombotic co-prescription. Current guidelines are therefore based on expert opinion and the extrapolation of non-randomised data. Patients can develop independent indications for antiplatelet and oral anticoagulant therapy, but in most cases the pathophysiology will intersect. The relationship between cardiovascular disease and atrial fibrillation is the typical example, where one fifth of patients presenting with atrial fibrillation will subsequently require coronary intervention, and up to one fifth of acute coronary syndrome (including ST elevation myocardial infarction, non-ST elevation myocardial infarction, and unstable angina) presentations develop atrial …

Background: The PlA2 polymorphism of glycoprotein IIIa (GPIIIa) has been previously identified as being associated with myocardial infarction (MI), but whether this represents a true association is entirely unclear due to differences in findings from different studies. We performed a meta-analysis to evaluate whether this polymorphism is a risk factor for MI. Methods: Electronic databases (MEDLINE and EMBASE) were searched for all articles evaluating genetic polymorphisms of GPIIIa. For studies where acute coronary events were recorded in association with genetic analysis, pooled odds ratios (ORs) were calculated using fixed-effects and random-effects models. The primary outcome measure was MI, and a secondary analysis was also performed for acute coronary syndromes (ACS) more generally. Findings: 57 studies were eligible for statistical analysis and included 17,911 cases and 24,584 controls. Carriage of the PlA2 allele was significantly associated with MI (n = 40,692; OR 1.077, 95% CI 1.024–1.132; p = 0.004) but with significant publication bias (p = 0.040). The degree of association with MI increased with decreasing age of subjects (≤45 years old: n = 9,547; OR 1.205, 95% CI 1.067–1.360; p = 0.003) and with adjustment of data for conventional cardiovascular risk factors (n = 12,001; OR 1.240, 95% CI 1.117–1.376; p>0.001). There was a low probability of publication bias for these subgroup analyses (all p>0.05). Conclusions: The presence of significant publication bias makes it unclear whether the association between carriage of the PlA2 allele and MI is true for the total population studied. However for younger subjects, the relative absence of conventional cardiovascular risk factors results in a significant association between carriage of the PlA2 allele and MI.

Abstract —Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet β-adrenoceptors (βARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of l -[ 3 H]-arginine to l -[ 3 H]-citrulline, after βAR stimulation or cAMP elevation. Basal NOS activity was 0.11±0.03 pmol l -citrulline/10 8 platelets. The βAR agonist isoproterenol 1 μmol/L and the adenylyl cyclase activator forskolin 1 μmol/L each increased NOS activity, to 0.26±0.04 and 0.23±0.03 pmol l -citrulline/10 8 platelets, respectively ( P 2+ . In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3±0.2% versus 2.6±0.2% respectively; P 2 ARs, as determined by coincubation with βAR subtype-selective antagonists. We conclude that β 2 ARs activate platelet NOS by increasing cAMP, and that this activation is Ca 2+ -independent. β 2 ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the l -arginine/NO system mediates the effects of β 2 ARs on adhesion but not aggregation. ( Circ Res. 2000;87:39-44.)

Some studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. We wished to determine the contribution of the L-arginine / NO system to vasodilatation in response to beta-adrenoceptor stimulation with isoprenaline or cyclic adenosine-3',5'-monophosphate (cyclic AMP) elevation with forskolin and dibutyryl cyclic AMP in vivo, using a rabbit femoral artery constant perfusion model. Baseline femoral artery pressure was similar in rabbits receiving isoprenaline, forskolin or dibutyryl cyclic AMP. Isoprenaline, forskolin and dibutyryl cyclic AMP each decreased femoral artery pressure in a dose-dependent manner. The doses (mol kg(-1)) of isoprenaline, forskolin and dibutyryl cyclic AMP which decreased pressure by 10% from baseline, expressed as a negative logarithm (-log ED(10)) were: 10.0+/-0.2, 9.5+/-0.1 and 4.9+/-0.1 respectively (P

Background We tested the hypothesis that direct renin inhibition with aliskiren protects against myocardial ischemia/reperfusion (I/R) injury in spontaneously hypertensive rats ( SHR ), and examined the mechanism by which this occurs. Methods and Results Male SHR were treated (orally, 4 weeks) with saline or aliskiren (30 or 60 mg kg −1 day −1 ) and subjected to 30 minutes of left anterior descending coronary artery occlusion followed by 6 or 24 hours of reperfusion. Only the higher dose significantly lowered systolic blood pressure, the lower dose causing a smaller apparent lowering that was nonsignificant. Despite this difference in blood pressure‐lowering effect, both doses increased the ejection fraction and fractional shortening and reduced myocardial infarct size equally. I/R decreased cardiac expression of phosphatidylinositol 3‐kinase (PI3K), phospho‐Akt and phospho‐endothelial nitric oxide synthase (phospho‐ eNOS ), but increased expression of inducible nitric oxide synthase ( iNOS ); these changes were all abrogated by aliskiren. Moreover, aliskiren decreased superoxide anion generation and increased cyclic guanosine‐3′,5′‐monophosphate, an index of bioactive nitric oxide, in myocardium. It also decreased the expression of myocardial matrix metalloproteinase‐2, matrix metalloproteinase‐9, and tissue inhibitor of metalloproteinases‐1 ( TIMP ‐1) following I/R. In a L angendorff heart preparation, the detrimental cardiac effects of I/R were abrogated by aliskiren, and these protective effects were abolished by NOS or PI3K inhibition. In a parallel study, although specific iNOS inhibition reduced plasma malondialdehyde and myocardial superoxide anion generation, it did not affect the deleterious effects of I/R on myocardial structure and function. Conclusions Direct renin inhibition protects against myocardial I/R injury through activation of the PI3K‐Akt‐ eNOS pathway.

BackgroundThe PlA1/A2 polymorphism of glycoprotein IIIa (GPIIIa) has been reported to be associated with risk of stroke in some studies, although other studies suggest no such association. This meta-analysis and systematic review was conducted to investigate the hypothesis that carriage of the PlA2 allele is a risk factor for stroke.MethodsElectronic databases (MEDLINE and EMBASE) were searched for all articles evaluating carriage of the PlA2 allele and the incidence of stroke. Pooled odds ratios (ORs) were calculated using fixed-effect and random-effect models.FindingsA total of 35 articles were eligible for inclusion, of which 25 studies were suitable for statistical analysis. For carriage of the PlA2 allele, OR 1.12 (n = 11,873; 95% CI = 1.03-1.22; p = 0.011) was observed for the incidence of stroke in adults, with subgroup analyses identifying the association driven by stroke of an ischaemic (n = 10,494; OR = 1.15, 95% CI = 1.05-1.27; p = 0.003) but not haemorrhagic aetiology (n = 2,470; OR = 0.90, 95% CI = 0.71-1.14; p = 0.398). This association with ischaemic stroke was strongest in individuals homozygous for the PlA2 allele compared to those homozygous for wild-type PlA1 (n = 5,906; OR = 1.74, 95% CI = 1.34-2.26; p0.05).ConclusionsThe totality of published data supports the hypothesis that carriage of the PlA2 polymorphism of GPIIIa is a risk factor for ischaemic strokes, and specifically those of cardioembolic and large vessel origin.

Background Endothelial dysfunction promotes atherosclerosis. We investigated whether in vivo magnetic resonance imaging ( MRI ) using an albumin‐binding contrast agent, gadofosveset, could monitor the efficacy of minocycline and ebselen in reducing endothelial permeability and atherosclerotic burden in the brachiocephalic artery of high‐fat diet ( HFD )–fed ApoE −/− mice. Methods and Results ApoE −/− mice were scanned 12 weeks after commencement of either a normal diet (controls) or an HFD . HFD ‐fed ApoE −/− mice were either untreated or treated with minocycline or ebselen for 12 weeks. Delayed‐enhancement MRI and T 1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R 1 , s −1 ) in untreated HFD ‐fed ApoE −/− mice (R 1 =3.8±0.52 s −1 ) compared with controls (R 1 =2.15±0.34 s −1 , P 1 =2.7±0.17 s −1 , P 1 =2.7±0.23 s −1 , P −/− mice showed less vessel wall enhancement compared with untreated HFD ‐fed ApoE −/− mice. Mass spectroscopy showed a lower gadolinium concentration in the brachiocephalic artery of treated (minocycline=28.5±3 μmol/L, ebselen=32.4±4 μmol/L) compared with untreated HFD ‐fed ApoE −/− mice (191±4.8 μmol/L) ( P MRI (minocycline=0.14±0.02 mm 2 , ebselen=0.20±0.09 mm 2 , untreated=0.44±0.01 mm 2 ; P 2 , ebselen=0.18±0.02 mm 2 , untreated=0.32±0.04 mm 2 ; P P P =0.03), with significant attenuation of the proinflammatory Ly6C high subtype (untreated mice, 42.64±6.1% of total monocytes; ebselen, 14.07±9.5% of total monocytes; minocycline, 26.42±0.6% of total monocytes). Conclusions We demonstrate that contrast‐enhanced MRI with an albumin‐binding contrast agent can be used to noninvasively monitor the effect of interventions on endothelial permeability and plaque burden. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim at restoring endothelial integrity.

Background Acute exercise may exert deleterious effects on the cardiovascular system through a variety of pathophysiological mechanisms, including increased platelet activation. However, the degree of exercise-induced platelet activation in untreated hypertensive (UH) individuals as compared with normotensive (NT) individuals has yet to be established. Furthermore, the effect of antihypertensive treatment on exercise-induced platelet activation in essential hypertension (EH) remains unknown. Methods Study 1 consisted of 30 UH and 15 NT subjects. UH subjects who received treatment were included in study 2 and were followed-up after a 3-month treatment period with an angiotensin II receptor blocker (ARB; valsartan). Circulating monocyte-platelet aggregates (MPA) and platelet P-selectin were measured as platelet activation markers at baseline, immediately after a treadmill exercise test, and 10, 30, and 90 minutes later. Results Maximal platelet activation was observed at 10 minutes after peak exercise in both groups. In UH subjects, MPA levels remained increased at 30 minutes after peak exercise, despite BP fall to baseline levels. MPA levels were significantly higher in UH subjects than NT subjects at maximal exercise and at 10 and 30 minutes of recovery. Post-treatment MPA levels increased significantly only at 10 minutes into recovery and were similar to those of NT subjects. Conclusions Acute high-intensity exercise exaggerates platelet activation in untreated patients with EH compared with NT individuals. Angiotensin II receptor blockade with adequate BP control greatly improves exercise-induced platelet activation in EH. Further studies are needed to clarify whether this phenomenon depends purely on BP lowering or benefits also from the pleiotropic effects of ARBs.

The American and European Societies of Cardiology have recently issued updated guidelines for the management of ST-elevation myocardial infarction (STEMI) and non-STEMI/unstable angina (NSTEMI/UA). The distinction between these two clinical manifestations of acute coronary syndrome (ACS) was first introduced in the 1996 publication of the “ACC/AHA Guidelines for the Management of Patients with Acute Myocardial Infarction”, when the term ACS was used to describe an episode of cardiac discomfort/pain arising from atherosclerotic plaque disruption. Patients suffering an ACS were categorized into STEMI and NSTEMI/UA on the basis of electrocardiography (ECG), to distinguish between those with likely complete thrombotic occlusion of a coronary artery leading to ST-elevation (STEMI patients) and those with putatively incomplete coronary artery occlusion in whom raised markers of myocardial necrosis would further distinguish myocardial infarction without ST-elevation (NSTEMI) from UA. Almost concomitantly, the European Society of Cardiology (ESC) published the guidelines for “Acute myocardial infarction: pre-hospital and in-hospital management” where a similar distinction was made between STEMI and non-STEMI-ACS.

Hypertension is a common disease associated with important cardiovascular complications. Persistent blood pressure of 140/90 or higher despite combined use of a reninangiotensin system blocker, calcium channel blocker and a diuretic at highest tolerated doses constitutes resistant hypertension. Excess sympathetic activity plays an important pathogenic role in resistant hypertension in addition to contributing to the development of metabolic problems, in particular diabetes. Reduction of renal sympathetic activity by percutaneous catheter-based radiofrequency ablation via the renal arteries has been shown in several studies to decrease blood pressure in patients with resistant hypertension, and importantly is largely free of significant complications. However, longer term follow-up is required to confirm both long-term safety and efficacy.

Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10–200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

1. MK-462 (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H- indol-3-yl]ethylamine) is a novel selective 5-HT1D-receptor agonist which in clinical trials has been shown to be an effective antimigraine agent. As angiographic studies have shown that sumatriptan (an established 5-HT1D-receptor agonist) can cause coronary artery vasoconstriction in patients, we compared the effects of MK-462 with those of 5-HT and those of sumatriptan, on isolated segments of human coronary artery in vitro. 2. Coronary arteries were obtained from explanted hearts from patients (n = 22, 2 females, 20 males, aged 21-60 years) undergoing cardiac transplantation. Endothelium-denuded ring segments of coronary artery, 2mm long were mounted in organbaths for isometric tension recording. For each arterial ring segment, a cumulative concentration-effect curve to either 5-HT, sumatriptan or MK-462 was determined. After maximal response to each agonist had been obtained, ketanserin (a 5-HT2 receptor antagonist) 0.6 microM was added to the tissue bath, followed by methiotepin (0.6 microM) and the reduction in tension produced by the addition of each antagonist was determined. 3. Out of 22 coronary arteries studied, only 10 showed any response (contraction) to 5-HT. Not all arteries which responded to 5-HT contracted in response to both sumatriptan and MK-462 (one ring from each artery being exposed to a single agonist in each case).(ABSTRACT TRUNCATED AT 250 WORDS)