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Until recently, it has been generally held that stable angina pectoris (SAP) primarily reflects the presence of epicardial coronary artery stenoses due to atheromatous plaque(s), while acute myocardial infarction (AMI) results from thrombus formation on ruptured plaques. This concept is now challenged, especially by results of the ORBITA and ISCHEMIA trials, which showed that angioplasty/stenting does not substantially relieve SAP symptoms or prevent AMI or death in such patients. These disappointing outcomes serve to redirect attention towards anomalies of small coronary physiology. Recent studies suggest that coronary microvasculature is often both structurally and physiologically abnormal irrespective of the presence or absence of large coronary artery stenoses. Structural remodelling of the coronary microvasculature appears to be induced primarily by inflammation initiated by mast cell, platelet, and neutrophil activation, leading to erosion of the endothelial glycocalyx. This leads to the disruption of laminar flow and the facilitation of endothelial platelet interaction. Glycocalyx shedding has been implicated in the pathophysiology of coronary artery spasm, cardiovascular ageing, AMI, and viral vasculitis. Physiological dysfunction is closely linked to structural remodelling and occurs in most patients with myocardial ischemia, irrespective of the presence or absence of large-vessel stenoses. Dysfunction includes the impairment of platelet and vascular responsiveness to autocidal coronary vasodilators, such as nitric oxide, prostacyclin, and hydrogen sulphide, and predisposes both to coronary vasoconstriction and to a propensity for microthrombus formation. These findings emphasise the need for new directions in medical therapeutics for patients with SAP, as well as a wide range of other cardiovascular disorders.

Impairment of the nitric oxide/soluble guanylate cyclase (NO)/sGC) signalling cascade is associated with many forms of cardiovascular disease, resulting not only in compromised vasodilatation but also loss of anti-aggregatory homeostasis. Myocardial ischaemia, heart failure, and atrial fibrillation are associated with moderate impairment of NO/sGC signalling, and we have recently demonstrated that coronary artery spasm (CAS) is engendered by severe impairment of platelet NO/sGC activity resulting in combined platelet and vascular endothelial damage. We therefore sought to determine whether sGC stimulators or activators might normalise NO/sGC homeostasis in platelets. ADP-induced platelet aggregation and its inhibition by the NO donor sodium nitroprusside (SNP), the sGC stimulator riociguat (RIO), and the sCG activator cinaciguat (CINA) alone or in addition to SNP were quantitated. Three groups of individuals were compared: normal subjects ( n = 9), patients (Group 1) with myocardial ischaemia, heart failure and/or atrial fibrillation ( n = 30), and patients (Group 2) in the chronic stage of CAS ( n = 16). As expected, responses to SNP were impaired ( p = 0.02) in patients versus normal subjects, with Group 2 patients most severely affected ( p = 0.005). RIO alone exerted no anti-aggregatory effects but potentiated responses to SNP to a similar extent irrespective of baseline SNP response. CINA exerted only intrinsic anti-aggregatory effects, but the extent of these varied directly (r = 0.54; p = 0.0009) with individual responses to SNP. Thus, both RIO and CINA tend to normalise anti-aggregatory function in patients in whom NO/sGC signalling is impaired. The anti-aggregatory effects of RIO consist entirely of potentiation of NO, which is not selective of platelet NO resistance. However, the intrinsic anti-aggregatory effects of CINA are most marked in individuals with initially normal NO/sGC signalling, and thus their magnitude is at variance with extent of physiological impairment. These data suggest that RIO and other sGC stimulators should be evaluated for clinical utility in both prophylaxis and treatment of CAS.

Perhexiline (Px) inhibits carnitine palmitoyltransferase 1 (CPT1), which controls uptake of long chain fatty acids into mitochondria. However, occasional cases of hypoglycaemia have been reported in Px-treated patients, raising the possibility that Px may also increase sensitivity to insulin. Furthermore, Px increases anti-aggregatory responses to nitric oxide (NO), an effect which may theoretically parallel insulin sensitization. We therefore sought to examine these relationships in patients with stable Type 2 diabetes (T2D) and cardiovascular disease (n = 30). Px was initiated, and dosage was titrated, to reach the therapeutic range and thus prevent toxicity. Investigations were performed before and after 2 weeks, to examine changes in insulin sensitivity and, utilizing aggregometry in whole blood, platelet responsiveness to the anti-aggregatory effects of the NO donor sodium nitroprusside (SNP). Other parameters that affect may affect NO signalling were also evaluated. Px substantially potentiated inhibition of platelet aggregation by SNP (from 16.7 ± 3.0 to 27.3 ± 3.7%; p = 0.005). Px did not change fasting blood glucose concentrations but reduced insulin sensitivity (HOMA-IR score increased from median of 4.47 to 6.08; p = 0.028), and increased fasting plasma insulin concentrations (median 16.5 to 19.0 mU/L; p = 0.014). Increases in SNP responses tended (r = -0.30; p = 0.11) to be reciprocally related to increases in HOMA-IR, and increases in HOMA-IR were greater ( p = 0.002) in patients without NO-sensitizing effects. No patient developed symptomatic hypoglycaemia, nor was there any other short-term toxicity of Px. Thus, in patients with stable T2D and cardiovascular disease, Px increases anti-aggregatory responsiveness to NO, but is not an insulin sensitizer, and does not induce hypoglycaemia. Absence of NO-sensitizing effect occurs in approximately 30% of Px-treated patients with T2D, and is associated with induction of insulin resistance in these patients.

Since its initial description by Japanese investigators 30 years ago, TakoTsubo Syndrome (TTS) has variously been regarded as a form of acute coronary syndrome and also as a form of cardiomyopathy (or more accurately, a myocarditis). There is actually good evidence that TTS embodies both of these concepts, and the main purpose of this review is to present data that they occur sequentially. The initial phase of the disorder (over perhaps the first 48 hours post onset of symptoms) represents a form of vasculitis, with associated damage to the endothelial glycocalyx and associated permeabilization of blood vessels. This is followed by a more prolonged phase of myocardial inflammation and oedema, associated with inflammatory activation and energetic impairment within the entire myocardium. Although this phase subsides after several months, it may be followed by longstanding impairment of myocardial function, reflecting residual fibrosis. Understanding of this gradual transition in TTS pathogenesis from vasculature towards myocardium remains an important limitation of patient management, especially as many patients are still told that their hearts have "recovered" within 1-2 weeks. A number of important uncertainties remain. These include development of specific early and ongoing therapeutic strategies to be used to match the sequential pathogenesis of TTS. "And so these men of Indostan Disputed loud and long, Each in his own opinion Exceeding stiff and strong, Though each was partly in the right, And all were in the wrong!" From: Six wise men of Hindustan., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2022 The Author(s). Published by IMR Press.)

The propensity towards platelet-rich thrombus formation increases substantially during normal ageing, and this trend is mediated by decreases in platelet responsiveness to the anti-aggregatory nitric oxide (NO) and prostacyclin (PGI 2 ) pathways. The impairment of soluble guanylate cyclase and adenylate cyclase-based signalling that is associated with oxidative stress represents the major mechanism of this loss of anti-aggregatory reactivity. Platelet desensitization to these autacoids represents an adverse prognostic marker in patients with ischemic heart disease and may contribute to increased thrombo-embolic risk in patients with heart failure. Patients with platelet resistance to PGI 2 also are unresponsive to ADP receptor antagonist therapy. Apart from ischemia, diabetes and aortic valve disease are also associated with impaired anti-aggregatory homeostasis. This review examines the association of impaired platelet cyclic nucleotide (i.e., cGMP and cAMP) signalling with the emerging evidence of thromboembolic risk in cardiovascular diseases, and discusses the potential therapeutic strategies targeting this abnormality.

Introduction: Since Prinzmetal first described a 'variant' form of angina pectoris, with predominantly resting episodes of pain and cyclic severity variations, it has gradually become apparent that this clinical presentation is caused by episodes of coronary artery spasm (CAS) involving focal or diffuse changes in large and/or small coronary arteries in the presence or absence of 'fixed' coronary artery stenoses. However, most clinicians have only limited understanding of this group of disorders., Areas Covered: We examine the clinical presentation of CAS, associated pathologies outside the coronary vasculature, impediments to making the diagnosis, provocative diagnostic tests, available and emerging treatments, and the current understanding of pathogenesis., Expert Opinion: CAS is often debilitating and substantially under-diagnosed and occur mainly in women. Many patients presenting with CAS crises have non-diagnostic ECGs and normal serum troponin concentrations, but CAS can be suspected on the basis of history and association with migraine, Raynaud's phenomenon and Kounis syndrome. Definitive diagnosis requires provocative testing at coronary angiography. Treatment still centers around the use of calcium antagonists, but with greater understanding of pathogenesis, new management options are emerging.

Background and Purpose: The pathophysiology of coronary artery spasm (CAS), with its associated ischaemic crises, is currently poorly understood and treatment is frequently ineffective. In view of increasing evidence that platelet-based defects may occur in CAS patients, we investigated platelet reactivity in CAS patients and whether symptomatic crises reflect activation of platelet-endothelial interactions., Experimental Approach: CAS patients were evaluated during acute and/or chronic symptomatic phases and compared with healthy control subjects. Inhibition of ADP-induced platelet aggregation by the NO donor sodium nitroprusside (SNP) and plasma concentrations of syndecan 1 (glycocalyx shedding marker), tryptase (mast cell activation marker) and platelet microparticles were measured., Key Results: Inhibition of platelet aggregation by SNP was diminished in chronic CAS, with further (non-significant) deterioration during symptomatic crises, whereas plasma concentrations of syndecan 1, tryptase and platelet microparticles increased. Treatment of patients with high-dose N-acetylcysteine (NAC) plus glyceryl trinitrate rapidly increased platelet responsiveness to SNP and decreased plasma syndecan 1 concentrations. The effect of NAC on platelet responsiveness to SNP was confirmed in vitro and mimicked by the H 2 S donor NaHS. Conversely, inhibition of enzymatic production of H 2 S attenuated NAC effect., Conclusion and Implications: CAS is associated with substantial impairment of platelet NO signalling. During acute symptomatic exacerbations, platelet resistance to NO is aggravated, together with mast cell activation and damage to both vasculature and platelets. NAC, via release of H 2 S, reverses platelet resistance to NO and terminates glycocalyx shedding during symptomatic crises: This suggests that H 2 S donors may correct the pathophysiological anomalies underlying CAS., (© 2021 The British Pharmacological Society.)

It is now 30 years since Japanese investigators first described Takotsubo Syndrome (TTS) as a disorder occurring mainly in ageing women, ascribing it to the impact of multivessel coronary artery spasm. During the intervening period, it has become clear that TTS involves relatively transient vascular injury, followed by prolonged myocardial inflammatory and eventually fibrotic changes. Hence symptomatic recovery is generally slow, currently an under-recognised issue. It appears that TTS is induced by aberrant post-β 2 -adrenoceptor signalling in the setting of "surge" release of catecholamines. Resultant activation of nitric oxide synthases and increased inflammatory vascular permeation lead to prolonged myocardial infiltration with macrophages and associated oedema formation. Initially, the diagnosis of TTS was made via exclusion of relevant coronary artery stenoses, plus the presence of regional left ventricular hypokinesis. However, detection of extensive myocardial oedema on cardiac MRI imaging offers a specific basis for diagnosis. No adequate methods are yet available for definitive diagnosis of TTS at hospital presentation. Other major challenges remaining in this area include understanding of the recently demonstrated association between TTS and antecedent cancer, the development of effective treatments to reduce risk of short-term (generally due to shock) and long-term mortality, and also to accelerate symptomatic recovery., (Copyright © 2020. Published by Elsevier B.V.)

Introduction: On-treatment platelet aggregability represents the major form of functional assessment for patients treated with P2Y 12 receptor antagonists, with "high" on-treatment platelet aggregability (HTPA) predicting thrombotic risk. However HTPA reflects a variable combination of pre-treatment hyperaggregability and poor response to P2Y 12 antagonists. We have previously shown that integrity of platelet adenylate cyclase/cAMP signaling, assessed with PGE 1 , is a strong predictor of individual responses to clopidogrel. We therefore sought to determine the extent to which HTPA reflects impaired platelet responsiveness to clopidogrel., Methods: Using data from our previous investigations of acute and sub-acute effects of clopidogrel, we analyzed the relationship between on-treatment aggregability and acute/steady state responsiveness to clopidogrel, utilizing ADP, the thromboxane A 2 mimetic U46619, and thrombin receptor-activating peptide (TRAP) as pro-aggregants. The relationship between anti-aggregatory response to PGE 1 and both on-treatment and pre-treatment aggregability was also examined., Results and Conclusions: With all 3 pro-aggregants, (1) response to clopidogrel after 4 h, as measured by ΔADP response, exhibits a strong inverse relationship with on-treatment aggregation, with a similarly inverse relationship between pre-treatment PGE 1 response and on-treatment aggregability; (2) there is a weaker inverse relationship between clopidogrel response and pre-treatment platelet aggregability, and a significant inverse relationship between pre-treatment PGE 1 response and pre-treatment platelet aggregability. Furthermore, pre-treatment PGE 1 response also predicts on-treatment platelet aggregability in response to ADP at steady state. Thus, HTPA largely represents clopidogrel resistance., (Copyright © 2020. Published by Elsevier Ltd.)

To determine (a) whether chronic heart failure with reduced ejection fraction (HFrEF) is associated with increased glycocalyx shedding; (b) whether glycocalyx shedding in HFrEF with left ventricular dyssynchrony is related to inflammation, endothelial dysfunction and/or redox stress and is ameliorated by cardiac resynchronisation therapy. Glycocalyx shedding has been reported to be increased in heart failure and is a marker of increased mortality. Its role in dyssynchronous systolic heart failure and the effects of cardiac resynchronisation therapy (CRT) are largely unknown. Twenty-six patients with dyssynchronous HFrEF were evaluated before and 6 months after CRT insertion. Echocardiographic septal to posterior wall delay (SPWD) assessed intra-ventricular mechanical dyssynchrony, and quality of life, integrity of nitric oxide (NO) signalling, inflammatory and redox-related biomarkers were measured. Glycocalyx shedding was quantitated via plasma levels of the glycocalyx component, syndecan-1. Syndecan-1 levels pre-CRT were inversely correlated with LVEF (r = - 0.45, p = 0.02) and directly with SPWD (r = 0.44, p = 0.02), QOL (r = 0.39, p = 0.04), plasma NT-proBNP (r = 0.43, p = 0.02), and the inflammatory marker, symmetric dimethylarginine (SDMA) (r = 0.54, p = 0.003). On multivariate analysis, syndecan-1 levels were predicted by SPWD and SDMA (β = 0.42, p = 0.009 and β = 0.54, p = 0.001, respectively). No significant correlation was found between syndecan-1 levels and other markers of endothelial dysfunction/inflammatory activation. Following CRT there was no significant change in syndecan-1 levels. In patients with dyssynchronous HFrEF, markers of glycocalyx shedding are associated with the magnitude of mechanical dyssynchrony and elevation of SDMA levels and inversely with LVEF. However, CRT does not reverse this process.

Introduction: P2Y 12 receptor antagonists reduce risk of thrombotic complications after stent implantation but increase bleeding risk. Activation of P2Y 12 receptors by ADP causes Gi-protein-mediated inhibition of adenylate cyclase (AC), thus limiting platelet response to anti-aggregatory effect of prostacyclin (PGI 2 ). However, P2Y 12 blockade reverses this ADP-induced suppression of the platelet PGI 2 /AC signaling pathway. We previously demonstrated that impairment of this pathway predicts poor response to clopidogrel., Objectives: To identify clinical correlates of variability in PGI 2 /AC signaling, and to assess the impact of such variability on individual responses to the direct P2Y 12 receptor antagonists ticagrelor (in vivo) and 2-methyl-thioadenosine-monophosphate (2MeSAMP) (in vitro)., Patients/methods: We compared the inhibitory effects of prostaglandin E 1 (PGE 1 ) and the PGI 2 analog Iloprost (Ilt) on platelet aggregation in whole blood samples from healthy control subjects (n = 17), and patients with stable angina pectoris (SAP; n = 35) or acute coronary syndromes (ACS; n = 23), with or without associated diabetes/hyperglycemia., Results: Compared to control subjects, patients with ACS and - to a lesser extent - those with SAP, exhibited impaired responses to PGE 1 , accentuated in the presence of hyperglycemia. Efficacy of ticagrelor treatment, measured as change in platelet reactivity index, was directly related to pre-treatment PGE 1 response, both at univariate and multivariate analysis. There was a strong correlation between extent of inhibition of platelet aggregation, whether by PGE 1 or Ilt, and the anti-aggregatory effect of 2MeSAMP in vitro., Conclusions: The integrity of PGI 2 /AC signaling, which is impaired in the presence of ACS and hyperglycemia, predetermines the anti-aggregatory efficiency of P2Y 12 receptor antagonists., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Activation of neutrophils is a critically important component of the innate immune response to bacterial and chemical stimuli, and culminates in the "neutrophil burst", which facilitates neutrophil phagocytosis via the release of superoxide anion radical (O₂ - ) from NADPH oxidase. Excessive and/or prolonged neutrophil activation results in substantial tissue injury and increases in vascular permeability-resulting in sustained tissue infiltration with neutrophils and monocytes, and persistent vasomotor dysfunction. Cardiovascular examples of such changes include acute and chronic systolic and diastolic heart failure ("heart failure with preserved ejection fraction"), and the catecholamine-induced inflammatory disorder takotsubo syndrome. We have recently demonstrated that B-type natriuretic peptide (BNP), acting via inhibition of activation of neutrophil NADPH oxidase, is an important negative modulator of the "neutrophil burst", though its effectiveness in limiting tissue injury is partially lost in acute heart failure. The potential therapeutic implications of these findings, regarding the development of new means of treating both acute and chronic cardiac injury states, are discussed.

Aims: To evaluate whether peripheral circulatory 'remodelling' as measured by changes in vascular compliance and in markers of nitric oxide signalling contributes to patient response to cardiac resynchronization therapy (CRT)., Methods and Results: Effects of CRT were evaluated in 33 patients pre-procedure and 6 months post-procedure. Peak oxygen consumption, 6 min walk distance, New York Heart Association class, and quality of life score were evaluated. Augmentation index and its interactions with nitric oxide (NO) were evaluated by applanation tonometry. Platelet NO responsiveness and content of thioredoxin-interacting protein were assessed. Plasma concentrations of N-terminal proBNP, asymmetric and symmetric dimethylarginine (SDMA), high sensitivity C-reactive protein, catecholamines, and matrix metalloproteinases-2 and -9 were assessed. Despite significant improvement in 6 min walk distance (P = 0.005), New York Heart Association class (P < 0.001), quality of life (P = 0.001), and all echocardiographic parameters post-CRT, there were no significant changes in augmentation index measurements, thioredoxin-interacting protein content, and platelet NO response. Significant falls in N-terminal proBNP (P = 0.008) and SDMA (P = 0.013; independent of renal function) occurred. Falls in SDMA predicted reduction in high-sensitivity C-reactive protein (P = 0.04) and increases in peak oxygen consumption (P = 0.04). There were no correlations between changes in echocardiographic parameters and those in vascular function., Conclusions: These data suggest that the beneficial effects of CRT over 6 months are independent of any change in peripheral NO-related signalling. However, there is evidence that suppression of inflammation occurs, and its magnitude predicts extent of clinical improvement., (© 2017 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Altered platelet physiology may contribute to the emergence of thrombosis in patients with many forms of cardiovascular disease. Excess platelet activation may reflect increased stimulation of pro-aggregatory pathways. There is, however, increasing evidence that excessive platelet response, due to impaired efficacy of anti-aggregatory autacoids such as nitric oxide (NO) and prostacyclin (PGI 2 ), may be just as important. For example, diminished platelet response to NO has been documented in acute and chronic myocardial ischaemia, heart failure, aortic valve disease and in the presence of hyperglycaemia. This "NO resistance" has been shown to reflect both the scavenging of NO by reactive oxygen species and dysfunction of its intracellular "receptor", soluble guanylate cyclase. Importantly, these abnormalities of NO signalling are potentially reversible through judicious application of pharmacotherapy. The analogous condition of impaired PGI 2 /adenylate cyclase (AC) signalling has received comparatively less attention to date. We have shown that platelet response to prostaglandin E 1 (PGE 1 ) is frequently impaired in patients with symptomatic myocardial ischaemia. Because the effects of ADP receptor antagonists such as clopidogrel and ticagrelor at the level of the P2Y 12 receptor are coupled with changes in activity of AC, impaired response to PGE 1 might imply both increased thrombotic risk and a reduced efficacy of anti-aggregatory drugs. Accordingly, patient response to treatment with clopidogrel is determined not only by variability of clopidogrel bio-activation, but also extensively by the integrity of platelet AC signalling. We here review these recent developments and their emerging therapeutic implications for thrombotic disorders.

Background: Female patients with atrial fibrillation (AF) experience increased risk of thromboembolism compared to males, an observation that is reflected by its inclusion in the CHA2DS2VASc score. New onset AF (often associated with tachycardia) also confers upon patients increased thromboembolic risk. The mechanisms underlying this risk are uncertain, but new onset AF is associated with profound impairment of platelet nitric oxide (NO) signalling. Given that cardiovascular responses to catecholamines are gender-dependent, and that the presence of tachycardia in new onset AF may represent a response to catecholaminergic stimulation, we explored the potential impact of gender and tachycardia on platelet aggregation and NO signalling., Methods: Interactions were sought in 87 AF patients between the extent of adenosine diphosphate (ADP)-induced platelet aggregation, the anti-aggregatory effects of the NO donor, sodium nitroprusside, gender, and admission heart rate. The potential impact of platelet expression of thioredoxin-interacting protein (Txnip) was also evaluated., Results: Analysis of covariance confirmed the presence of physiological antagonism between platelet ADP and NO responses [F (1, 74) = 12.212, P < 0.01], while female sex correlated with impaired NO responses independent of platelet aggregability [F (2, 74) = 8.313, P < 0.01]. Admission heart rate correlated directly with platelet aggregation (r = 0.235, P < 0.05), and inversely with NO response (r = -0.331, P < 0.01). Txnip expression varied neither with gender nor with heart rate., Conclusions: These results indicate that gender and heart rate are independent determinants of platelet function. Prospective studies of the putative benefit of reversal of tachycardia on restoration of normal platelet function are therefore a priority.

Objective: Atrial fibrillation (AF) is a condition where platelet hyperaggregability is commonly present. We examined potential physiological bases for platelet hyperaggregability in a cohort of patients with acute and chronic AF. In particular, we sought to identify the impact of inflammation [myeloperoxidase (MPO) and C-reactive protein (CRP)] and impaired nitric oxide (NO) signaling., Methods: Clinical and biochemical determinants of adenosine diphosphate (ADP)-induced platelet aggregation were sought in patients (n = 106) hospitalized with AF via univariate and multivariate analysis., Results: Hyper-responsiveness of platelets to ADP was directly (r = 0.254, p < 0.01) correlated with plasma concentrations of thrombospondin-1 (TSP-1), a matricellular protein that impairs NO responses and contributes to development of oxidative stress. In turn, plasma TSP-1 concentrations were directly correlated with MPO concentrations (r = 0.221, p < 0.05), while MPO concentrations correlated with those of asymmetric dimethylarginine (ADMA, r = 0.220, p < 0.05), and its structural isomer symmetric dimethylarginine (SDMA, r = 0.192, p = 0.05). Multivariate analysis identified TSP-1 (β = 0.276, p < 0.05) concentrations, as well as female sex (β = 0.199, p < 0.05), as direct correlates of platelet aggregability, and SDMA concentrations (β = - 0.292, p < 0.05) as an inverse correlate., Conclusion: We conclude that platelet hyperaggregability, where present in the context of AF, may be engendered by impaired availability of NO, as well as via MPO-related inflammatory activation.

Background: Risk of substantial haemorrhage represents a critically important limitation to effective anti-thrombotic treatment in patients with atrial fibrillation (AF). While it is known that this risk is increased in anticoagulated patients either in the presence of anti-aggregatory drugs or concomitant renal insufficiency, there are currently few data on the potential interactions between endogenous platelet aggregability and bleeding risk., Objective: We therefore evaluated in a cohort of AF patients: (1), the putative relationship between platelet aggregability and HAS-BLED score; (2), the potential biochemical bases for such a relationship., Methods: Patients were included as part of SAFETY, a randomised controlled trial evaluating outpatient management of AF patients. Platelet response to ADP was evaluated via whole blood impedance aggregometry; clinical and biochemical correlates of platelet aggregation were sought via univariate and multivariate analysis., Results: Platelet aggregation correlated inversely (r=-0.220, p<0.05) with HAS-BLED score. Univariate biochemical correlates of decreased platelet aggregation were plasma concentrations of symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). On multivariate analyses, plasma SDMA concentration (β=-0.318, p<0.01), platelet content of thioredoxin-interacting protein (Txnip, β=0.261, p<0.05) and plasma thrombospondin-1 (TSP-1, β=0.249, p<0.05) concentration were predictive of platelet ADP response. Consistent with previous reports, plasma SDMA concentrations were strongly and inversely correlated with estimated glomerular filtration rate (eGFR, r=-0.780, p<0.001)., Conclusions: These data therefore suggest that (1), physiologically impaired, like pharmacologically impaired, platelet aggregability may increase bleeding risk in anticoagulated AF patients; (2), the biochemical basis for this may include impaired effects of nitric oxide (via Txnip, TSP-1) but also concomitant renal dysfunction.

Background: and, Hypotheses: The signal transduction pathway modulated by activation or blockade of platelet P2Y12 receptors is linked to PGE1-stimulated adenylate cyclase effects, but this link's impact on P2Y12 receptor antagonist response is uncertain. We therefore tested the hypothesis that pre-treatment platelet responsiveness to PGE1 predicts subsequent responsiveness to clopidogrel., Methods: In order to maximise heterogeneity of platelet responsiveness to PGE1 we investigated both healthy subjects (n=30) and patients with CHD undergoing elective coronary stenting (n=22), all genotyped for common CYP2C19 variants associated with clopidogrel sensitivity (CS). We determined baseline pre-clopidogrel platelet sensitivity to the inhibitory effects of PGE1 by ADP-induced whole blood aggregation. Clopidogrel was administered for 7days utilising a weight-based regimen. CS was expressed as change (Δ) in ADP-induced aggregation and in VASP-phosphorylation (VASP-P). We used univariate and multivariate analysis to correlate such parameters with PGE1 sensitivity, BMI and presence/absence of CHD., Results: In the study cohort, pre-treatment responsiveness to PGE1 varied widely (70±28 [standard deviation (SD)]% inhibition of aggregation: range 10 to 100%). In the entire study cohort, pre-treatment PGE1 sensitivity correlated with CS irrespective of genotype. On univariate analysis, CS was not significantly greater for patients without than those with loss-of-function mutations. Moreover, at multivariate analysis, PGE1 sensitivity, but not genotype, was a strong correlate of ΔADP and ΔVASP-P (P<0.0001 for both)., Conclusions: The integrity of the cAMP pathway is a major determinant of subacute CS., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Aims: The release of the B-type natriuretic peptide (BNP) is increased in heart failure (HF), a condition associated with oxidative stress. BNP is known to exert anti-inflammatory effects including suppression of neutrophil superoxide (O2(-)) release. However, BNP-based restoration of homeostasis in HF is inadequate, and the equivocal clinical benefit of a recombinant BNP, nesiritide, raises the possibility of attenuated response to BNP. We therefore tested the hypothesis that BNP-induced suppression of neutrophil O2(-) generation is impaired in patients with acute HF., Methods and Results: We have recently characterized suppression of neutrophil O2(-) generation (PMA- or fMLP-stimulated neutrophil burst) by BNP as a measure of its physiological activity. In the present study, BNP response was compared in neutrophils of healthy subjects (n = 29) and HF patients (n = 45). Effects of BNP on fMLP-induced phosphorylation of the NAD(P)H oxidase subunit p47phox were also evaluated. In acute HF patients, the suppressing effect of BNP (1 µmol/L) on O2(-) generation was attenuated relative to that in healthy subjects (P < 0.05 for both PMA and fMLP). Analogously, BNP inhibited p47phox phosphorylation in healthy subjects but not in HF patients (P < 0.05). However, O2(-)-suppressing effects of the cell-permeable cGMP analogue (8-pCPT-cGMP) were preserved in acute HF. Conventional HF treatment for 5 weeks partially restored neutrophil BNP responsiveness (n = 25, P < 0.05), despite no significant decrease in plasma NT-proBNP levels., Conclusions: BNP inhibits neutrophil O2(-) generation by suppressing NAD(P)H oxidase assembly. This effect is impaired in acute HF patients, with partial recovery during treatment., (© 2015 The Authors. European Journal of Heart Failure © 2015 European Society of Cardiology.)

Background: Hyperglycemia in patients with acute coronary syndromes is associated with poor outcomes, and its rapid correction with insulin infusion has been shown to restore platelet responsiveness to nitric oxide and to suppress superoxide (O2(-)) generation. Thioredoxin-interacting protein has emerged recently as a pivotal modulator of hyperglycemia-induced inflammation, O2(-) production, and impairment of nitric oxide signaling, but it is not known whether its expression in platelets can be downregulated rapidly., Methods: In 12 hyperglycemic patients with acute coronary syndrome, we evaluated the putative role of thioredoxin-interacting protein suppression in the platelet nitric oxide response after reversal of hyperglycemia with insulin infusion., Results: Insulin infusion for 13.0 ± 0.8 (standard error of the mean) hours decreased blood glucose level from 16.6 ± 1.6 mmol/L to 8.7 ± 1.4 mmol/L (P = .002). This induced (1) sensitization of antiaggregatory response to nitric oxide (from 6.5% ± 7.7% to 39.7% ± 7.0%, P < .0001); (2) improved endothelial progenitor cell function (from a median of 45 to 180 colony-forming units, P < .05); and (3) decreases of whole blood reactive oxygen species content (P < .05). However, there was no significant suppression of platelet thioredoxin-interacting protein expression (mean decrease, 59 arbitrary units; 95% confidence interval, -193 to +74)., Conclusions: Correction of hyperglycemia in patients with acute coronary syndrome rapidly reverses oxidative stress, restoring both platelet nitric oxide responsiveness and endothelial progenitor cell function, but this process is largely or entirely independent of thioredoxin-interacting protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Clopidogrel resistance is more common in patients with loss-of-function CYP2C19 genotypes. Since adenylate cyclase (AC) and soluble guanylate cyclase (sGC) pathways are variably impaired in patients with ischaemic heart disease, we tested the relevance of these determinants in patients undergoing acute loading with clopidogrel (600 mg) prior to non-emergent coronary stenting., Methods: Inhibitory effects of prostaglandin E1 (PGE1, an AC activator) and sodium nitroprusside (NP, a sGC activator) on platelet aggregation were determined at baseline and compared with platelet responses to clopidogrel (4 h after administration) assessed as ∆ADP, and Platelet Reactivity Index (∆PRI). Data were analysed according to CYP2C19 genotype., Results: In patients without loss of function mutations (n=18), ∆ADP but not ∆PRI, was directly correlated with baseline PGE1 responsiveness (rs=0.62, p=0.005)). NP responsiveness did not predict ∆ADP. However there was no relationship between clopidogrel responses and either PGE1 or NP responsiveness in patients with loss of function mutations. Multivariate correlates of clopidogrel response were both the genotype status (β=-0.609, p<0.001) and the baseline response to PGE1 (β=0.303, p=0.03)., Conclusions: While genetically impaired bio-activation markedly limits acute (4 h) clopidogrel response, impaired AC signalling provides an additional cause for clopidogrel resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Clinical factors associated with thromboembolic risk in AF patients are well characterized and include new onset AF. Biochemically, AF is associated with inflammatory activation and impairment of nitric oxide (NO) signalling, which may also predispose to thromboembolism: the bases for variability in these anomalies have not been identified. We therefore sought to identify correlates of impaired platelet NO signalling in patients hospitalized with atrial fibrillation (AF), and to evaluate the impact of acuity of AF., Methods: 87 patients hospitalized with AF were evaluated. Platelet aggregation, and its inhibition by the NO donor sodium nitroprusside, was evaluated using whole blood impedance aggregometry. Correlates of impaired NO response were examined and repeated in a "validation" cohort of acute cardiac illnesses., Results: Whilst clinical risk scores were not significantly correlated with integrity of NO signalling, new onset AF was associated with impaired NO response (6 ± 5% inhibition versus 25 ± 4% inhibition for chronic AF, p<0.01). New onset AF was a multivariate correlate (p<0.01) of impaired NO signalling, along with platelet ADP response (p<0.001), whereas the associated tachycardia was not. Platelet ADP response was predicted by elevation of plasma thrombospondin-1 concentrations (p<0.01). Validation cohort evaluations confirmed that acute AF was associated with significant (p<0.05) impairment of platelet NO response, and that neither acute heart failure nor acute coronary syndromes were associated with similar impairment., Conclusion: Recent onset of AF is associated with marked impairment of platelet NO response. These findings may contribute to thromboembolic risk in such patients., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Many acute cardiovascular disease states are associated with neutrophil infiltration of myocardium and subsequent release of superoxide (O2 (-) ) and myeloperoxidase (MPO), which contribute to inflammatory reactions. B-Type natriuretic peptide (BNP) is known to exert anti-inflammatory and antifibrotic effects, but it is not known whether these may include interactions with neutrophils. In neutrophils isolated from 20 healthy subjects, we assessed the effect of BNP on the 'neutrophil burst' (O2 (-) production and MPO release) stimulated by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively. Effects of BNP on cGMP accumulation, and the effects of the cell-permeable cGMP analogue 8-(4-chlorophenylthio) guanosine-cGMP (8-p-CPT-cGMP) and protein kinase G (PKG) inhibition with KT5823 on the neutrophil-BNP interaction were also evaluated. B-Type natriuretic peptide suppressed O2 (-) release from neutrophils by 23 ± 6% (P < 0.001) and 24 ± 8% (P < 0.05) following PMA and fMLP stimulation, respectively. Although BNP did not significantly increase cGMP formation, 8-p-CPT-cGMP suppressed both PMA- and fMLP-induced neutrophil O2 (-) release by 16% and 28%, respectively (P < 0.05). The PKG inhibitor KT5823 attenuated the effects of BNP on both fMLP- and PMA-associated O2 (-) production. Neither BNP nor 8-p-CPT-cGMP significantly affected MPO release from neutrophils. Suppression of O2 (-) release from neutrophils by BNP may contribute to its anti-inflammatory and antifibrotic actions., (© 2014 Wiley Publishing Asia Pty Ltd.)

The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Previous studies in non-human blood vessels and in platelets have demonstrated that under hypoxic conditions release of NO from nitrite (NO2(-)) is potentiated by deoxyhaemoglobin. In the current study, we characterized hypoxic potentiation of NO2(-) effects in human vasculature and platelets in vitro, addressing underlying mechanisms. The vasodilator efficacy of NO2(-), in comparison with glyceryl trinitrate (GTN), was evaluated in vitro, using segments of human saphenous vein. Under hypoxic conditions, there was a leftward shift of the NO2(-) concentration-response curve (EC50: 22 μM in hyperoxia vs 3.5 μM in hypoxia; p<0.01), but no significant potentiation of GTN effect. In the presence of red blood cells, hypoxic potentiation of NO2(-) vasodilator effect was accentuated. In whole blood samples and platelet-rich plasma (PRP) we assessed inhibition of platelet aggregation by NO2(-) (1mM), in comparison with that of sodium nitroprusside (SNP, 10 μM). In individual subjects (n=37), there was a strong correlation (r=0.75, p<0.0001) between anti-aggregatory effects of NO2(-) and SNP in whole blood, signifying that resultant sGC activation underlies biological effect and responses to NO2(-) are diminished in the presence of NO resistance. In PRP, the effects of NO2(-) were less pronounced than in whole blood (p=0.0001), suggesting an important role of Hb (within RBCs) in the bioconversion of NO2(-) to NO. Inhibition of platelet aggregation by NO2(-) was almost 3-fold greater in venous than in arterial blood (p<0.0001), and deoxyHb concentration directly correlated (r=0.69, p=0.013) with anti-aggregatory response. Incremental hypoxia applied to venous blood samples (in hypoxic chamber) caused a progressive increase in both deoxyHb level and anti-aggregatory effect of NO2(-). When subjects inhaled a 12% O2 mixture for 20 min, there was a 3-fold rise in blood deoxyHb fraction (p<0.01). In PRP, response to NO2(-) also increased under hypoxia, and was further enhanced (p<0.01) by deoxyHb. Furthermore, deoxyHb exerted significant anti-aggregatory effects even in the absence of added NO2(-), suggesting a role for endogenous NO2(-). The results of this work provide further mechanistic insights into hypoxic potentiation of vasodilator and anti-aggregatory actions of NO2(-). In human saphenous veins and blood, the balance of evidence suggests differential rates of NO release from NO2(-) (largely modulated by deoxyHb) as the fundamental mechanism., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Background: Impaired generation and signaling of nitric oxide (NO) contribute substantially to cardiovascular (CV) risk (CVR) associated with hypertension, hyperlipidemia, and diabetes mellitus. In our rapidly aging society, advanced age is, in itself, a consistent and independent CVR factor. Many processes involved in aging are modulated by NO. We therefore postulated that aging might be independently associated with impaired NO signaling., Methods and Results: In a prospective cohort study of 204 subjects (mean age 63±6 at study entry), we evaluated the effects of 4 years of aging on parameters of NO generation and effect, including platelet aggregability and responsiveness to NO, and plasma concentrations of the NO synthase inhibitor, asymmetric dimethylarginine (ADMA). Clinical history, lipid profile, high-sensitivity C-reactive protein, routine biochemistry, and 25-hydroxyvitamin D levels were obtained at study entry and after 4 years of follow-up. Aging was associated with marked deterioration of responsiveness of platelets to NO (P<0.0001) and increases in plasma ADMA concentrations (P<0.0001). There was a significant correlation between changes in these parameters over time (r=0.2; P=0.013). On multivariable analyses, the independent correlates of deterioration of responsiveness of platelets to NO were female gender (β=0.17; P=0.034) and low vitamin D concentrations (β=0.16; P=0.04), whereas increases in ADMA were associated with presence of diabetes (β=0.16; P=0.03) and impaired renal function (β=0.2; P=0.004)., Conclusions: Aging is associated with marked impairment of determinants of NO generation and effect, to an extent which is commensurate with adverse impact on CV outcomes. This deterioration represents a potential target for therapeutic interventions., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

The thioredoxin system, which consists of thioredoxin (Trx), nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), has emerged as a major anti-oxidant involved in the maintenance of cellular physiology and survival. Dysregulation in this system has been associated with metabolic, cardiovascular, and malignant disorders. Thioredoxin-interacting protein (TXNIP), also known as vitamin D-upregulated protein or thioredoxin-binding-protein-2, functions as a physiological inhibitor of Trx, and pathological suppression of Trx by TXNIP has been demonstrated in diabetes and cardiovascular diseases. Furthermore, TXNIP effects are partially Trx-independent; these include direct activation of inflammation and inhibition of glucose uptake. Many of the effects of TXNIP are initiated by its dissociation from intra-nuclear binding with Trx or other SH-containing proteins: these effects include its migration to cytoplasm, modulating stress responses in mitochondria and endoplasmic reticulum, and also potentially activating apoptotic pathways. TXNIP also interacts with the nitric oxide (NO) signaling system, with apparent suppression of NO effect. TXNIP production is modulated by redox stress, glucose levels, hypoxia and several inflammatory activators. In recent studies, it has been shown that therapeutic agents including insulin, metformin, angiotensin converting enzyme inhibitors and calcium channel blockers reduce TXNIP expression, although it is uncertain to what extent TXNIP suppression contributes to their clinical efficacy. This review addresses the role of TXNIP in health and in cardiovascular and metabolic disorders. Finally, the potential advantages (and disadvantages) of pharmacological suppression of TXNIP in cardiovascular disease and diabetes are summarized.

Objectives: Bicuspid aortic valve (BAV) is associated with increased risk of valvular degeneration and ascending aortic aneurysm formation and rupture. We sought to evaluate the roles of endothelial dysfunction and inflammatory activation in modulating these processes., Methods: We performed a case-control study of patients with BAV together with a multivariate analysis within the BAV group to identify factors associated with: development of significant valvular disease; dilatation of the ascending aorta; differential valve relative to aortic disease. Endothelial function of patients and controls was evaluated via flow-mediated dilatation (FMD) and plasma concentrations of asymmetric dimethylarginine (ADMA). Correlations with inflammatory markers and endothelial progenitor cell counts were also examined. Morphological and physiological assessment of the valve and ascending aorta was performed with transthoracic echocardiography and MRI., Results: Patients with BAV (n=43) and controls (n=25) were matched for age and gender. FMD was significantly lower in patients than controls (7.85±3.48% vs 11.58±3.98%, p=0.001), and these differences were age-independent. Within the BAV cohort, multivariate correlates of peak aortic valve velocity were plasma concentrations of ADMA and myeloperoxidase (MPO) (both p<0.01), while increasing age was an independent correlate of ascending aortic diameter (p<0.05). Furthermore, both low FMD and inflammatory activation were multivariate correlates of selectivity for valvular disease., Conclusions: BAV is associated with endothelial dysfunction. The extent of inflammatory activation (specifically MPO release) and that of endothelial dysfunction impact primarily on integrity of the valve rather than aortic structure.

Purpose: Little information is available concerning the mechanism(s) underlying Takotsubo cardiomyopathy (TTC), other than evidence of associated catecholamine secretion. Given the known effects of catecholamines on endothelial function, we tested the hypothesis that TTC might also be associated with impairment of nitric oxide (NO) signaling. We now report an evaluation of NO signaling in TTC patients (vs. aged-matched controls) in relation to (a) severity of the acute attack and (b) rate of recovery., Methods: In 56 patients with TTC, we utilized (1) platelet responsiveness to NO and (2) plasma levels of asymmetric dimethylarginine (ADMA) as indices of integrity of the cyclic guanosine monophosphate (cGMP) pathway. Additionally, endothelial progenitor cell (EPC) counts, which are partially NO-dependent, were evaluated. These parameters were measured at the time of diagnosis and 3 months thereafter, and compared with an aging female cohort (n = 81)., Results: The data suggested that both NO generation and effect were accentuated in TTC patients: ADMA concentrations were lower (p = 0.003), and responsiveness to NO substantially greater (p = 0.0001) than in controls both acutely and after 3 months. Markers of severity of TTC attacks directly correlated with NO responsiveness, while extent of recovery at 3 months varied inversely with ADMA concentrations., Conclusion: TTC is associated with intensification of NO signaling relative to that in normal age-matched females. Our data are consistent with this intensified signal's potential contribution to the extent of initial myocardial injury, but conversely to accelerated recovery.

Impaired platelet responsiveness to nitric oxide (NO resistance) is a common characteristic of many cardiovascular disease states and represents an independent risk factor for cardiac events and mortality. NO resistance reflects both scavenging of NO by superoxide (O2(-)), and impairment of the NO receptor, soluble guanylate cyclase (sGC). There is thus an urgent need for circumvention of NO resistance in order to improve clinical outcomes. Nitroxyl (HNO), like NO, produces vasodilator and anti-aggregatory effects, largely via sGC activation, but is not inactivated by O2(-). We tested the hypothesis that HNO circumvents NO resistance in human platelets. In 57 subjects with or without ischemic heart disease, platelet responses to the HNO donor isopropylamine NONOate (IPA/NO) and the NO donor sodium nitroprusside (SNP) were compared. While SNP (10μM) induced 29±3% (p<0.001) inhibition of platelet aggregation, IPA/NO (10μM) caused 75±4% inhibition (p<0.001). In NO-resistant subjects (n=28), the IPA/NO:SNP response ratio was markedly increased (p<0.01), consistent with partial circumvention of NO resistance. Similarly, cGMP accumulation in platelets was greater (p<0.001) with IPA/NO than with SNP stimulation. The NO scavenger carboxy-PTIO (CPTIO, 200μM) inhibited SNP and IPA/NO responses by 92±7% and 17±4% respectively (p<0.001 for differential inhibition), suggesting that effects of IPA/NO are only partially NO-mediated. ODQ (10μM) inhibited IPA/NO responses by 36±8% (p<0.001), consistent with a contribution of sGC/haem to IPA/NO inhibition of aggregation. There was no significant relationship between whole blood ROS content and IPA/NO responses. Thus the HNO donor IPA/NO substantially circumvents platelet NO resistance while acting, at least partially, as a haem-mediated sGC activator., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

The clinical efficacy of the P2Y12 receptor antagonist clopidogrel as an agent to prevent thrombotic events predominantly reflects its anti-aggregatory effects. Stent thrombosis in particular is more likely to occur in patients in whom clopidogrel effect is limited. "Resistance" to clopidogrel in general should theoretically arise either because of a reduction in plasma concentration of the active metabolite and/or of the downstream intracellular biochemical changes underlying antiplatelet effects. We therefore postulate that "resistance" to clopidogrel arises via a combination of pharmacogenetic, pharmacokinetic and intracellular biochemical mechanisms. Considerable attention has been so far directed to the finding that stent thrombosis occurs more frequently in patients with loss-of-function mutations of CYP2C19, thus limiting clopidogrel bioactivation. Furthermore, a number of drug-drug interactions may marginally impair responsiveness to clopidogrel, largely via impairment of bioactivation. However, population data also suggest that clopidogrel "resistance" occurs more frequently in patients with acute coronary syndromes than in normal subjects, and that "resistance" is particularly common in obese subjects and with diabetes. Here we critically review available literature and speculate on the possibility that non-genetic causes of clopidogrel "resistance" may arise from impairments of the intracellular signaling cascade initiated by P2Y12 receptor inhibition. In such cases, "resistance" to clopidogrel may also theoretically occur with other P2Y12 receptor antagonists, irrespective of the need for bioactivation. Delineation of this non-genetic component of "resistance" to P2Y12 inhibitors may facilitate the development of optimal therapeutic strategies for high-risk cardiovascular patients., (© 2013.)

Background: Impaired tissue responsiveness to nitric oxide (NO) occurs in many cardiovascular diseases as well as with advanced age and is a correlate of poor outcomes. This phenomenon results from oxidative stress, with NO "scavenging" and dysfunction of soluble guanylate cyclase (sGC). Thioredoxin-interacting protein (TXNIP) is a major intracellular regulator of inflammatory activation and redox stress, but its interactions with NO/sGC are poorly understood. We have now evaluated the relationship between platelet TXNIP expression and function of the NO/sGC axis in subjects of varying age and during therapy with ramipril., Methods & Results: Young (n=42) and aging (n=49) subjects underwent evaluation of platelet TXNIP content. Aging subjects additionally had measurements of platelet NO responsiveness and routine biochemistry. Platelet TXNIP content was greater (376±33 units) in the aging compared to younger subjects (289±13 units; p<0.05). In the aging subjects there was a significant negative correlation (r=-0.50, p<0.001) between platelet TXNIP content and NO responsiveness. In a separate cohort of 15 subjects two week treatment with ramipril, which reversed platelet NO resistance and potentiated sGC activity, also decreased platelet TXNIP content by 40% (p=0.011)., Conclusions: Platelet TXNIP content increases with aging, varies inversely with responsiveness to NO, and diminishes rapidly following treatment with ramipril. These data suggest that TXNIP-induced oxidative stress may be a critical modulator of tissue resistance to NO, a fundamental basis for cardiovascular disease. Analogously suppression of TXNIP expression can potentially be utilized as an index of restoration of cardiovascular homeostasis., (© 2013.)

Objective: The objective of this study was to compare the impact of aging on nitric oxide (NO) modulation of platelet and vascular function in healthy women and women with polycystic ovary syndrome., Methods and Results: A case-control study of women ages 18 to 60 years, comparing women with polycystic ovarian syndrome against age-matched healthy controls, was performed. A total of 242 women, of whom 109 had polycystic ovarian syndrome (based on Rotterdam criteria), participated in the study. Women who were pregnant or on clopidogrel were excluded from the study. Inhibition of platelet aggregation by nitric oxide (primary outcome measure), vascular endothelial function, plasma concentrations of N(G), N(G)-dimethyl-L-arginine (ADMA), endothelial progenitor cell count, and high-sensitivity C-reactive protein (markers of endothelial dysfunction and inflammation) were assessed. With increasing age in control women, there was progressive attenuation of platelet responses to NO, impairment of endothelial function, and elevation of ADMA levels (P ≤.001). Irrespective of age, women with polycystic ovarian syndrome exhibited greater impairment of all these parameters (all P <.05, 2-way analysis of variance) and demonstrated these anomalies earlier in life., Conclusions: Normal aging in women is associated with attenuation of NO-based signaling in platelets and blood vessels. In women with polycystic ovarian syndrome, these changes are present from early adult life and may contribute to premature atherogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: Aortic valve stenosis (AS) and its precursor, aortic valve sclerosis (ASc), occur frequently in Western populations. Investigations to retard the progression of AS using statins have been unsuccessful. Development of ASc in humans is associated with increased aortic valve backscatter (AVBS) and poor tissue nitric oxide (NO) responsiveness. In an animal model, ramipril retarded AS/ASc development. We have now set out to identify factors associated with the progression of ASc in humans., Methods and Results: At baseline and after 4 years, 204 randomly selected subjects (age 63 ± 6 years at study entry) underwent echocardiography with the determination of AVBS values, measurements of platelet NO responsiveness, plasma asymmetric dimethylarginine concentrations, lipid profile, high-sensitivity-C-reactive protein, routine biochemistry, and 25-hydroxy-vitamin D levels. During the study period, 68% of subjects had detectable AVBS progression. On multivariate analysis, higher calcium concentrations (β = 0.22; P = 0.004), poor platelet NO responsiveness (β = 0.18; P = 0.018), and increased arterial stiffness (β = 0.15; P = 0.044) were independent predictors of disease progression. The use of angiotensin-converting enzyme-inhibitors/angiotensin II receptor blockers (ACE-I/ARB) predicted the lack of disease progression (assessed categorically) in the overall cohort and in those without ASc at baseline (n = 159) (β = 0.8; P = 0.025 and β = 1.3; P = 0.001, respectively). No conventional coronary risk factors were associated with disease progression., Conclusion: This study of early aortic valve disease (i) demonstrates that disease progression occurs in the majority of the normal ageing population over a 4-year period; (ii) provides evidence of the importance of the NO signalling cascade in disease development and progression; and (iii) provides additional data linking ACE-I/ARB use with the retardation of ASc.

Objectives: Using 2 sequential studies in HOPE (Heart Outcomes Prevention Evaluation) study-type patients, the aims of this study were: 1) to test the hypothesis that ramipril improves platelet nitric oxide (NO) responsiveness: and 2) to explore biochemical and physiological effects of ramipril in a cohort selected on the basis of platelet NO resistance., Background: Ramipril prevents cardiovascular events, but the bases for these effects remain uncertain. NO resistance at both the platelet and vascular levels is present in a substantial proportion of patients with diabetes or ischemic heart disease and is an independent risk factor for cardiovascular events., Methods: Study 1 was a double-blind, randomized comparison of ramipril (10 mg) with placebo in a cohort of patients (n = 119) with ischemic heart disease or diabetes plus additional coronary risk factor(s), in which effects on platelet responsiveness to NO were compared. Study 2 was a subsequent short-term evaluation of the effects of ramipril in a cohort of subjects (n = 19) with impaired platelet NO responsiveness in whom additional mechanistic data were sought., Results: In study 1, ramipril therapy increased platelet responsiveness to NO relative to the extent of aggregation (p < 0.001), but this effect occurred primarily in patients with severely impaired baseline NO responsiveness (n = 41). In study 2, ramipril also improved platelet NO responsiveness (p < 0.01), and this improvement was correlated directly with increased NO-stimulated platelet generation of cyclic guanosine monophosphate (p < 0.02) but not with changes in plasma thrombospondin-1 levels., Conclusions: Ramipril ameliorates platelet NO resistance in HOPE study-type patients, with associated increases in soluble guanylate cyclase responsiveness to NO. This effect is likely to contribute to treatment benefit and define patients in whom ramipril therapy is particularly effective., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Takotsubo cardiomyopathy (TTC) is a form of reversible acute cardiac dysfunction of uncertain pathogenesis, which occurs predominantly in postmenopausal women, often with antecedent severe stress. Systolic dysfunction most commonly affects the apex of the left ventricle. There is considerable uncertainty regarding the pathogenesis of TTC and the optimal diagnostic methodology. Acute catecholamine release may play a component role, but the regional hypokinesis is associated with an acute inflammatory process, with resultant early release of brain natriuretic peptide (BNP) and N-terminal pro-BNP. As the diagnosis of TTC has largely been a process of exclusion, there has been considerable underdiagnosis. The combination of demographics, preceding history, ECG appearances and N-terminal pro-BNP elevation may provide the basis for improved early diagnosis. Complete recovery takes at least several months, with a risk of recurrent episodes. Efforts to delineate pathogenesis, expedite diagnosis and evaluate residual disability may assist in the development of appropriate treatment regimens.

Background: Polycystic ovary syndrome (PCOS) is associated with incremental risk of atherosclerosis and possibly of cardiovascular events. Insulin resistance (IR) occurs frequently in PCOS subjects, which might be one of the mechanisms involved in engendering such risk. We sought to evaluate whether the impact of other factors potentially associated both with PCOS and with IR might differentially modulate degree of IR in women with and without PCOS., Methods and Results: We measured body mass index (BMI), hs-CRP, plasma concentrations of asymmetric dimethylarginine (ADMA), vitamin D (25(OH)D3) levels and platelet responsiveness to nitric oxide donor sodium nitroprusside (NO responsiveness) in 47 young women (n=27 with PCOS and n=20 weight-matched controls) without metabolic syndrome, hypertension or overt cardiovascular disease. We performed univariate and multivariate regression analyses to establish correlates of the quantitative insulin-sensitivity check index (QUICKI), as a marker of IR. On univariate analysis, plasma 25(OH)D3 levels and low NO responsiveness tended to be direct correlates with QUICKI in the entire subject group. BMI, hs-CRP, and ADMA levels were significant inverse correlates of QUICKI in PCOS subjects, but not in subjects without PCOS. On multivariate analysis, NO responsiveness, and 25(OH)D3 levels, but not PCOS per se were significant correlates of QUICKI., Conclusions: In the entire cohort of young women, low NO responsiveness and vitamin D deficiency are associated with low QUICKI, while elevated ADMA, inflammatory activation and obesity are selectively associated with low QUICKI in PCOS subjects; this may contribute to the increased cardiovascular risk associated with this syndrome., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aortic valve stenosis (AS) is the commonest form of valvular heart disease in the Western world. Its prevalence increases exponentially with age and it is present in 2-7% of all patients over 65 years of age. In view of the considerable cardiovascular morbidity and mortality associated not only with AS, but even its earlier stage, aortic sclerosis, many investigations have been directed towards better understanding of its pathogenesis, with the ultimate objective of developing strategies to retard its progression. Although risk factors and downstream mediators appear similar for AS and atherosclerosis (older age, male sex, hypertension, smoking, hypercholesterolemia, and diabetes, as many as 50% of patients with AS do not have clinically significant atherosclerosis. On the basis both of recent experimental evidence and clinical trials, it appears that atherogenesis is not pivotal to the pathogenesis of AS. On the other hand, there is increasing evidence of active involvement of aortic valve fibroblasts with resultant increased production of reactive oxygen species, active pro-inflammatory and pro-fibrotic processes culminating in calcification. We also discuss the evidence of involvement of the nitric oxide system in the pathogenesis of AS. The renin-angiotensin system has also emerged as a major player in the pathogenesis of AS. Histologically, there is increased ACE expression and elevated angiotensin II levels in stenotic valves, while we have just demonstrated amelioration of AS with the use of ACE inhibitors in an animal model. We further discuss intervention studies aimed at retarding AS progression, including recent failures of statins to retard progression of AS in large randomized clinical studies. Finally, we discuss the special case of bicuspid aortic valve, including its genetics and unique associated features.