Camilla Calvieri

Statins as Antithrombotic Drugs

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase.1 They are largely used in patients with or at risk of cardiovascular disease inasmuch as randomized trials have consistently shown that statins lower the rate of myocardial infarction, stroke, and cardiovascular death.2 The majority of trials with statins investigated patients with stable atherosclerotic disease, in whom the reduction in cardiovascular events appeared to be related to the cholesterol-lowering effect of statins and ultimately to plaque stabilization.3 However, experimental data demonstrated that statins may also exert a direct antithrombotic effect in models of arterial and venous thrombosis via a mechanism unrelated to the cholesterol-lowering activity4,5; this may suggest that statins inhibit several pathways of hemostasis, including platelet activation and coagulation cascade. Consistent with these observations are the potential negative properties that have been observed, including the association between statin therapy and cerebral hemorrhage. In this review, we present experimental data in support of the ability of statins to interfere directly with the clotting system and platelet activation, as well as the clinical settings that suggest that statins exert beneficial effects related to their antithrombotic properties. Colli et al6 were the first to show that statins interfere with activation of the clotting system and the coagulation cascade. Specifically, they demonstrated that fluvastatin dose-dependently inhibits activity of tissue factor (TF), a glycoprotein that converts factor X to factor Xa,7 and suggested that downexpression of geranylgeranylated protein is implicated in TF lowering. These data were confirmed in patients affected by polygenic hypercholesterolemia8 in whom simvastatin 20 mg/d for 8 weeks resulted in downregulation of TF antigen and activity and thrombin generation. Of note, inhibition of clotting activation, as assessed by plasma values of prothrombin fragment F1+2, a marker of …

NPR-C: a component of the natriuretic peptide family with implications in human diseases

The natriuretic peptide (NP) family includes atrial natriuretic peptide (ANP), B-type natriuretic peptide, C-type natriuretic peptide and their receptors NPR-A, NPR-B and NPR-C. The effects exerted by this hormonal system in the control of cardiovascular, renal and endocrine functions have been extensively investigated. Moreover, the involvement of NP in the pathogenesis of cardiovascular diseases has been demonstrated. Among the NP components, NPR-C has been described, at the time of its discovery, as the clearance receptor of NP devoid of any physiological functions. Emerging roles of NPR-C, however, have been highlighted over the last few years in relation to its effects on the cardiovascular system and other organs. These effects appear to be directly mediated through distinct cAMP-dependent intracellular mechanisms. Moreover, evidence has been accumulated on a potential pathophysiological role of NPR-C in human diseases. Ongoing studies from our group are revealing its involvement in the mediation of antiproliferative effects exerted on vascular cells by a molecular variant of human ANP. Thus, a new appraisal of NPR-C is overcoming the traditional view of a mere clearance receptor. This review focuses on the most important evidence supporting an involvement of NPR-C in mediating some of the actions of NP and its direct implication in cardiovascular diseases. The current state of knowledge highlights the need of further studies to better clarify the specific roles of NPR-C in pathophysiological processes.

Molecular mechanisms underlying cardiac antihypertrophic and antifibrotic effects of natriuretic peptides

Natriuretic peptides (NPs) exert well-characterized protective effects on the cardiovascular system, such as vasorelaxation, natri- and diuresis, increase of endothelial permeability, and inhibition of renin–angiotensin–aldosterone system. It has been reported that they also possess antihypertrophic and antifibrotic properties and contribute actively to cardiac remodeling. As a consequence, they are involved in several aspects of cardiovascular diseases. Antihypertrophic and antifibrotic actions of NPs appear to be mediated by specific signaling pathways within a more complex cellular network. Elucidation of the molecular mechanisms underlying the effects of NPs on cardiac remodeling represents an important research objective in order to gain more insights on the complex network leading to cardiac hypertrophy, ventricular dysfunction, and transition to heart failure, and in the attempt to develop novel therapeutic agents. The aim of the present article is to review well-characterized molecular mechanisms underlying the antihypertrophic and antifibrotic effects of NPs in the heart that appear to be mainly mediated by guanylyl cyclase type A receptor. In particular, we discuss the calcineurin/NFAT, the sodium exchanger NHE-1, and the TGFβ1/Smad signaling pathways. The role of guanylyl cyclase type B receptor, along with the emerging functional significance of natriuretic peptide receptor type C as mediators of CNP antihypertrophic and antifibrotic actions in the heart are also considered.

Lower mortality rate in elderly patients with community-onset pneumonia on treatment with aspirin.

Background Pneumonia is complicated by high rate of mortality and cardiovascular events (CVEs). The potential benefit of aspirin, which lowers platelet aggregation by inhibition of thromboxane A2 production, is still unclear. The aim of the study was to assess the impact of aspirin on mortality in patients with pneumonia. Methods and Results Consecutive patients admitted to the University‐Hospital Policlinico Umberto I (Rome, Italy) with community‐onset pneumonia were recruited and prospectively followed up until discharge or death. The primary end point was the occurrence of death up to 30 days after admission; the secondary end point was the intrahospital incidence of nonfatal myocardial infarction and ischemic stroke. One thousand and five patients (age, 74.7±15.1 years) were included in the study: 390 were receiving aspirin (100 mg/day) at the time of hospitalization, whereas 615 patients were aspirin free. During the follow‐up, 16.2% of patients died; among these, 19 (4.9%) were aspirin users and 144 (23.4%; P<0.001) were aspirin nonusers. Overall, nonfatal CVEs occurred in 7% of patients, 8.3% in nonaspirin users, and 4.9% in aspirin users (odds ratio, 1.77; 95% confidence interval, 1.03 to 3.04; P=0.040). The Cox regression analysis showed that pneumonia severity index (PSI), severe sepsis, pleural effusion, and PaO2/FiO2 ratio <300 negatively influenced survival, whereas aspirin therapy was associated with improved survival. Compared to patients receiving aspirin, the propensity score adjusted analysis confirmed that patients not taking aspirin had a hazard ratio of 2.07 (1.08 to 3.98; P=0.029) for total mortality. Conclusions This study shows that chronic aspirin use is associated with lower mortality rate within 30 days after hospital admission in a large cohort of patients with pneumonia.

C2238 Atrial Natriuretic Peptide Molecular Variant Is Associated With Endothelial Damage and Dysfunction Through Natriuretic Peptide Receptor C Signaling

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. Objective: We investigated the mechanisms underlying the vascular effects of C2238-&agr;ANP. Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-&agr;ANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-&agr;ANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-&agr;ANP reduced endothelial tube formation, as assessed by matrigel. C2238-&agr;ANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-&agr;ANP, as evaluated by intracellular cGMP levels. In contrast, C2238-&agr;ANP, but not T2238-&agr;ANP, markedly reduced intracellular cAMP levels in an NPR-C–dependent manner. Accordingly, C2238-&agr;ANP showed higher affinity binding to NPR-C, than T2238-&agr;ANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-&agr;ANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-&agr;ANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-&agr;ANP. Overall, these data indicate that C2238-&agr;ANP affects endothelial cell integrity through NPR-C–dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-&agr;ANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results. Conclusions: C2238-&agr;ANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

Pneumonia, thrombosis and vascular disease

An enhanced risk of cardiovascular mortality has been observed after pneumonia. Epidemiological studies have shown that respiratory tract infections are associated with an increased risk of thrombotic‐related vascular disease such as myocardial infarction, ischemic stroke and venous thrombosis. Myocardial infarction and stroke have been detected essentially in the early phase of the disease (i.e. within 48 h from hospital admission), with an incidence ranging from as low as 1% to as high as 11%. Age, previous cardiovascular events and high pneumonia severity index were independent predictors of myocardial infarction; clinical predictors of stroke were not identified. Deep venous thrombosis and pulmonary embolism may also occur after pneumonia but incidence and clinical predictors must be defined. The biological plausibility of such an association may be deduced by experimental and clinical studies, showing that lung infection is complicated by platelet aggregation and clotting system activation, as documented by up‐regulation of tissue factor and down‐regulation of activated protein C. The effect of antithrombotic drugs has been examined in experimental and clinical studies but results are still inconclusive.

Different behaviour of NOX2 activation in patients with paroxysmal/persistent or permanent atrial fibrillation

Background NOX2, the catalytic subunit of NADPH oxidase, is suggested to play a role in favouring the occurrence of atrial fibrillation (AF) after cardiac surgery via formation of reactive oxidant species. However, its role in spontaneous AF is still unclear. Objective To define the role of NOX2 and isoprostanes, a marker of oxidative stress, in the different settings of AF. Methods The study was performed on 174 patients with AF (82 with paroxysmal/persistent AF and 92 with permanent AF) and 90 controls matched for sex, age and atherosclerotic risk factors. Urinary isoprostanes and serum levels of soluble NOX2-derived peptide (sNOX2-dp) were measured in each patient. Results Urinary isoprostanes and sNOX2-dp concentrations were significantly higher in patients with paroxysmal/persistent AF than in those with permanent AF and controls. Compared with controls, patients with permanent AF showed a weak increase in sNOX2-dp and no difference in isoprostanes. Multivariable analyses demonstrated that baseline values of sNOX2-dp and urinary isoprostanes were independently associated with the type of AF (paroxysmal/persistent vs permanent; β=−224, p=0.007 and β=−231, p=0.005, respectively). A significant correlation between sNOX2-dp levels and urinary excretion of isoprostanes was also detected (R=0.707, p<0.001). Conclusions This study provides evidence that NOX2 is upregulated only in patients with paroxysmal/persistent AF and is responsible for overproduction of isoprostanes. This finding warrants further study to see if inhibition of NOX2 may reduce the risk of paroxysmal/persistent AF.

Atrial natriuretic peptide and regulation of vascular function in hypertension and heart failure: implications for novel therapeutic strategies.

Atrial natriuretic peptide (ANP) plays a pivotal role in modulation of vascular function and it is also involved in the pathophysiology of several cardiovascular diseases. We provide an updated overview of the current appraisal of ANP vascular effects in both animal models and humans. We describe the physiological implications of ANP vasomodulatory properties as well as the involvement of ANP, through its control of vascular function, in hypertension and heart failure. The principal molecular mechanisms underlying regulation of vascular tone, that is natriuretic peptide receptor type A/cyclic guanylate monophosphate, natriuretic peptide receptor type C, nitric oxide system, are discussed. We review the literature on therapeutic implications of ANP in hypertension and heart failure, examining the potential use of ANP analogues, neutral endopeptidase (NEP) inhibitors, ACE/NEP inhibitors, angiotensin receptor blocker (ARB)/NEP inhibitors, the new dual endothelin-converting enzyme (ECE)/NEP inhibitors and ANP-based gene therapy. The data discussed support the role of ANP in different pathological conditions through its vasomodulatory properties. They also indicate that ANP may represent an optimal therapeutic agent in cardiovascular diseases.

Nox2 up-regulation is associated with an enhanced risk of atrial fibrillation in patients with pneumonia

Background Community-acquired pneumonia (CAP) may be complicated by atrial fibrillation (AF) but the underlying mechanism is still unclear. Nox2-derived oxidative stress has been suggested to favour AF. Methods We consecutively enrolled 432 patients hospitalised for CAP. Nox2 activity, as assessed by serum levels of soluble Nox2 (sNox2-dp), was evaluated in each CAP patient. A 12-lead electrocardiography was repeated every 24 h. All patients were followed up until discharge. Results Forty-one patients with CAP (9.5%) experienced a new episode of AF within 24–72 h after hospital admission. Patients who experienced AF showed higher blood levels of sNox2-dp compared to those who did not (35.2±15.1 vs 27.0±12.5 pg/mL; p<0.001). Pneumonia Severity Index score (p=0.014), history of paroxysmal AF (p<0.001) and sNox2-dp (p=0.019) were independently associated with AF. At discharge, serum sNox2-dp levels were significantly decreased in the entire cohort (27.8±13.0 vs 21.9±6.8 pg/mL; p<0.001). Twenty-three out of 41 CAP patients with AF returned to sinus rhythm (56%); patients who remained in AF showed significantly higher baseline and discharge levels of sNox2-dp compared to those without AF (p<0.001) or with the 23 AF patients who returned to sinus rhythm (p<0.05). In vitro study showed that platelets or leucocytes incubated with endotoxin, at concentrations similar to those found in the circulation of CAP patients, elicited Nox2 up-regulation, suggesting endotoxin as a trigger of oxidative stress. Conclusions AF may be detected in the early phase of CAP and is associated with Nox2 activation, suggesting a role for oxidative stress in promoting this cardiac arrhythmia. Trial registration number NCT01773863.

Patients with microvascular obstruction after primary percutaneous coronary intervention show a gp91phox (NOX2) mediated persistent oxidative stress after reperfusion.

Background: Persistent oxidative stress may play a key role in microvascular obstruction (MVO). We aimed at assessing the role of platelet gp91phox (NOX2), the catalytic subunit of NADPH oxidase in MVO. Methods: We enrolled 40 patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention within 12 h from symptoms onset, either with angiographic MVO (n=20) or good angiographic myocardial reperfusion (MR) (n=20). Angiographic MVO was defined as a final thrombolysis in myocardial infarction (TIMI) flow ≤2 or TIMI flow of 3 with myocardial blush grade <2. NOX2 and isoprostanes (8-iso-PGF2α) levels, as assessed by enzyme-linked immunoadsorbent assay (ELISA) or by an enzyme immunoassays, respectively, were measured on admission, at 24 h and pre-discharge. Results: NOX2 levels increased from baseline to pre-discharge in patients with angiographic MVO (20.25 (15–24.75) pg/ml vs 25.50 (17–29.25) pg/ml, p=0.02), but not in MR patients (p=0.45), with a significant interaction between baseline and pre-discharge levels among the two groups (p=0.04). The levels of 8-iso-PGF2α showed a trend to increase from baseline to pre-discharge in angiographic MVO patients (295 (183.50–389.25) pmol/l vs 322 (206–370) pmol/l, p=0.06), but not in patients with MR (p=0.56), with a trend for interaction between baseline and pre-discharge levels among the two groups (p=0.09). Conclusion: Patients with MVO, but not those with myocardial reperfusion, have a sustained increase of NOX2 and 8-iso-PGF2α. Therapies targeting NOX2 or high dosage antioxidants should be tested for MVO prevention and treatment.