Giuseppe Di Stolfo

Pentaerythrityl Tetranitrate and Nitroglycerin, but not Isosorbide Mononitrate, Prevent Endothelial Dysfunction Induced by Ischemia and Reperfusion

Background—Short term exposure to nitroglycerin (GTN) has protective properties that are similar to ischemic preconditioning. Whether other organic nitrates such as pentaerithrityl tetranitrate (PETN) and isosorbide mononitrate (ISMN) have similar protective effects has not been explored. Methods and Results—In a randomized, parallel, double blind, controlled trial, 37 healthy young volunteers received no therapy (n=10), transdermal GTN 1.2 mg for 2 hours (n=9), PETN 80 mg (n=9), or ISMN 40 mg (n=9). Twenty-four hours later, endothelium-dependent flow-mediated vasodilation (FMD) was measured before and after local exposure to ischemia and reperfusion (IR). In the no therapy group, IR blunted FMD (FMD after IR: 1.9±0.6%, P<0.05), an effect that was prevented by GTN (FMD after IR: 5.3±1.4%, P<0.05 compared with no therapy). PETN had the same protective effect (FMD after IR: 8.1±1.3%, P<0.05 compared with no therapy), whereas ISMN had no significant pharmacological preconditioning effect (FMD after-IR: 3.6±0.8%, P=ns compared with no therapy). While it blocked the effect of GTN, Vitamin C (n=8) did not modify PETN preconditioning (FMD after IR: 6.3±0.9%, P=ns compared with before IR), showing that this phenomenon is not mediated by oxygen free radical production. In an effort to identify the mechanism of PETN preconditioning, isolated human endothelial cells were incubated with PETN, GTN, or ISMN. Only PETN induced expression of the genes encoding for heme oxygenase and ferritin, which have been involved in ischemic and pharmacological preconditioning. Conclusions—We show important differences among organic nitrates in their capacity to prevent IR-induced endothelial dysfunction. GTN and PETN, but not ISMN, have this preconditioning effect. The potential clinical implications of these data warrant further investigation.

Tolerance to nitroglycerin-induced preconditioning of the endothelium: a human in vivo study

Damage and dysfunction of the vascular endothelium critically influence clinical outcomes after ischemia and reperfusion (I/R). Brief exposure to organic nitrates can protect the vascular endothelium from I/R injury via a mechanism that is similar to ischemic preconditioning and is independent of hemodynamic changes. The clinical relevance of these protective effects clearly depends on whether they can be sustained over time. Twenty-four healthy (age 25-32) male volunteers were randomized to receive 1) transdermal nitroglycerin (GTN; 0.6 mg/h) administered for 2 h on 1 day only, 2) transdermal GTN for 2 h/day for 7 days, or 3) continuous therapy with transdermal GTN for 7 days. Eight volunteers underwent continuous GTN therapy followed by intra-arterial infusion of the antioxidant vitamin C. Finally, five additional subjects underwent no therapy and served as controls. Endothelial function measurements were performed before and after induction of I/R of the arm. I/R caused a significant blunting of the flow responses to acetylcholine in the control group (P < 0.01 vs. before I/R). A single 2-h GTN dosage, given 24 h before I/R, prevented I/R-induced endothelial dysfunction [P = not significant (NS) vs. before I/R], but this protective effect was completely lost after 1 wk of GTN administration 2 h/day (P < 0.05 vs. before I/R; P = NS vs. control). In subjects who received continuous GTN, endothelial responses were blunted before I/R, and I/R did not cause further endothelial dysfunction. Finally, vitamin C normalized acetylcholine responses and prevented the loss of preconditioning associated with prolonged GTN. In a separate experimental model using isolated human endothelial cells, short-term incubation with GTN caused upregulation of heme oxygenase, an effect that was lost after prolonged GTN administration. Although a single administration of GTN is able to protect the endothelium from I/R-induced endothelial dysfunction, this protection is lost upon prolonged exposure, likely via an oxidative mechanism.

Observations of time-based measures of flow-mediated dilation of forearm conduit arteries: implications for the accurate assessment of endothelial function.

Endothelium-dependent flow-mediated dilation (FMD) is measured as the increase in diameter of a conduit artery in response to reactive hyperemia, assessed either at a fixed time point [usually 60-s post-cuff deflation (FMD(60))] or as the maximal dilation during a 5-min continuous, ECG-gated, measurement (FMD(max-cont)). Preliminary evidence suggests that the time between reactive hyperemia and peak dilation (time to FMD(max)) may provide an additional index of endothelial health. We measured FMD(max-cont), FMD(60), and time to FMD(max) in 30 young healthy volunteers, 22 healthy middle-aged adults, 16 smokers, 23 patients with hypertension, 40 patients with coronary artery disease, and 22 patients with heart failure. As previously reported, FMD(max-cont) was similar in healthy cohorts and was significantly blunted in smokers and all patient groups, whereas FMD(60) was significantly blunted only in heart failure patients. There was a wide within-group variability between measures of time to FMD(max) with no significant difference between normal and patient groups. Intra-arterial infusion of the nitric oxide synthase inhibitor N(omega)-monomethyl-l-arginine in eight healthy subjects resulted in a blunting of FMD(max-cont) (P < 0.001) and FMD(60) (P = 0.02) but not time to FMD(max). Both FMD(max-cont) and FMD(60) demonstrated good repeatability in 30 young healthy volunteers studied on two separate occasions (P < 0.01 for both), whereas time to FMD(max) varied widely between visits (P = not significant). In conclusion, although time to FMD(max) does not appear to be a useful adjunctive measure of endothelial health, the use of continuous diameter measurements provides important data in the study of endothelial function in healthy subjects and patients with cardiovascular disease.

The ENPP1 Q121 Variant Predicts Major Cardiovascular Events in High-Risk Individuals: Evidence for Interaction With Obesity in Diabetic Patients

OBJECTIVE Insulin resistance (IR) and cardiovascular disease may share a common genetic background. We investigated the role of IR-associated ENPP1 K121Q polymorphism (rs1044498) on cardiovascular disease in high-risk individuals. RESEARCH DESIGN AND METHODS A prospective study (average follow-up, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80–2.70) in GHS, 2.31 (95% CI 1.22–4.34) in TVAS, and 1.36 (95% CI 0.88–2.10) in CREED, and 1.56 (95% CI 1.15–2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

Coadministration of Atorvastatin Prevents Nitroglycerin-Induced Endothelial Dysfunction and Nitrate Tolerance in Healthy Humans

OBJECTIVES We aimed to assess whether concurrent administration of atorvastatin would modify the development of tolerance and endothelial dysfunction associated with sustained nitroglycerin (GTN) therapy in humans. BACKGROUND Animal studies have demonstrated that administration of 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors can protect against GTN-induced endothelial dysfunction and tolerance, likely through an antioxidant mechanism. METHODS Thirty-six healthy male volunteers were randomized to receive continuous transdermal GTN (0.6 mg/h) and placebo, atorvastatin (80 mg/day) alone, or continuous transdermal GTN (0.6 mg/h) with concurrent atorvastatin (80 mg/day), all for 7 days. On the second visit, forearm blood flow was measured with venous-occlusion strain gauge plethysmography in response to incremental infusions of acetylcholine (7.5, 15, and 30 μg/min). Acetylcholine infusions were coinfused first with saline, and repeated during the coinfusion of vitamin C (24 mg/min). Blood pressure responses to sublingual GTN (400 μg) were assessed on both visits. RESULTS Acetylcholine responses in the GTN plus placebo group were significantly attenuated versus those in the GTN plus atorvastatin and atorvastatin groups (p < 0.01). Coinfusion of vitamin C completely restored acetylcholine responses in the GTN plus placebo group (p < 0.01 vs. saline coinfusion), but caused no change in either the atorvastatin or the GTN plus atorvastatin groups. Blood pressure responses to sublingual GTN did not significantly change between visits in subjects receiving GTN plus atorvastatin and atorvastatin alone, but were significantly blunted in the GTN plus placebo group (p < 0.05). CONCLUSIONS The present findings demonstrate, for the first time in humans, that atorvastatin prevents both GTN-induced endothelial dysfunction and nitrate tolerance, likely by counteracting the GTN-induced increase in oxidative stress.

Nitroglycerine causes mitochondrial reactive oxygen species production: In vitro mechanistic insights

BACKGROUND Nitroglycerine (GTN) is an organic nitrate that has been used for more than 100 years. Despite its widespread clinical use, several aspects of the pharmacology of GTN remain elusive. In a recent study, the authors of the present study showed that GTN causes opening of the mitochondrial permeability transition pore (mPTP) and mitochondrial production of reactive oxygen species (ROS). OBJECTIVE In the present study, it was tested whether GTN-induced ROS production depends on mitochondrial potassium ATP-dependent channel or mPTP opening, and/or GTN biotransformation. METHODS AND RESULTS Isolated rat heart mitochondria were incubated with succinate (a substrate for complex II) and GTN, causing immediate ROS production, as manifested by chemiluminescence. This ROS production was prevented by concomitant vitamin C incubation. Conversely, inhibitors of potassium ATP-dependent channels, mPTP opening or of GTN biotransformation did not modify ROS production. CONCLUSIONS GTN triggers mitochondrial ROS production independently of the opening of mitochondrial channels and/or of GTN biotransformation. The present data, coupled with previous evidence published by the same authors that GTN causes opening of mPTPs, provide further evidence on the pharmacology of GTN. It is proposed that GTN causes direct uncoupling of the respiratory chain, which determines ROS production and subsequent mPTP opening. The clinical implications of these findings are also discussed.

Folic Acid does not limit endothelial dysfunction induced by ischemia and reperfusion: a human study.

Nitric oxide synthase (NOS) uncoupling is a condition of increased production of superoxide anion associated with a decreased production of nitric oxide (NO) by this enzyme. Folic acid can prevent and/or reverse NOS uncoupling in the setting of diabetes, smoking, hypercholesterolemia, and nitrate tolerance. Whereas animal studies showed a protective effect of folic acid in ischemia and reperfusion (IR) injury, no study tested whether folic acid administration limits IR-induced endothelial dysfunction in humans. In a double-blind, parallel study, 20 healthy young male volunteers were randomized to receive folic acid, 10 mg/d for 7 days, or matching placebo. At the end of the treatment period, endothelium-dependent, flow-mediated dilation (FMD) of the radial artery was measured before and after IR injury (15 minutes of ischemia at the level of the brachial artery followed by 15 minutes of reperfusion). There was no difference at baseline between groups in any variable. In the placebo group, IR significantly blunted FMD (before IR, 6.7 ± 1.0%; after IR, 1.5 ± 1.3%, P < 0.01). A similar effect was observed in the folic acid group (before IR, 6.3 ± 1.1%; after IR, 2.1 ± 1.0%, P = ns compared with placebo). As opposed to animal studies, high-dose folic acid does not protect the vascular endothelium from IR injury in humans.

The mechanism of nitrate-induced preconditioning.

Nitroglycerin (GTN) has been shown, in both human and animal studies, to induce a protective phenotype that limits tissue damage after ischemia and reperfusion. This phenomenon is similar to ischemic preconditioning, and several reports suggest that also the molecular pathways involved in this protective effect of nitrates are the same that determine ischemic preconditioning. Our group conducted a series of studies aimed at investigating, using a human model of endothelial IR injury, the mechanism of nitrate-induced preconditioning and particularly the role of reactive oxygen species formation and of the opening of mitochondrial permeability transition pores. Our data demonstrate that GTN protects the endothelium against postischemic endothelial dysfunction in a mechanism that is mediated by oxygen free radical release and opening of mitochondrial permeability transition pores. In contrast, the protective effect of pentaerithrityl tetranitrate appears to be independent of these mechanisms, and it seems to be mediated by induction of antioxidant genes. Finally, isosorbide mononitrate seems to be devoid of a significant protective effect. These data are summarized and discussed in the present paper.

Serum uric acid as a prognostic marker in the setting of advanced vascular disease: A prospective study in the elderly

Background Many epidemiological studies analyze the relationship between hyperuricemia and cardiovascular outcomes. This observational prospective study investigates the association of serum uric acid (SUA) levels with adverse cardiovascular events and deaths in an elderly population affected by advanced atherosclerosis. Methods Two hundred and seventy six elderly patients affected by advanced atherosclerosis (217 males and 59 females; aged 71.2 ± 7.8 years) were included. All patients were assessed for history of cardiovascular disease, cancer, obesity and traditional risk factors. Patients were followed for approximately 31 ± 11 months. Major events were recorded during follow-up, defined as myocardial infarction, cerebral ischemia, myocardial and/or peripheral revascularization and death. Results Mean SUA level was 5.47 ± 1.43 mg/dL; then we further divided the population in two groups, according to the median value (5.36 mg/dL). During a median follow up of 31 months (5 to 49 months), 66 cardiovascular events, 9 fatal cardiovascular events and 14 cancer-related deaths have occurred. The patients with increased SUA level presented a higher significant incidence of total cardiovascular events (HR: 1.867, P = 0.014, 95% CI: 1.134–3.074). The same patients showed a significant increased risk of cancer-related death (HR: 4.335, P = 0.025, 95% CI: 1.204–15.606). Conclusions Increased SUA levels are independently and significantly associated with risk of cardiovascular events and cancer related death in a population of mainly elderly patients affected by peripheral vasculopathy.

‘B line’ in heart failure: a not so easy issue

We read with great interest the paper by Coiro et al.1 about the utilization of artefacts generated by chest ultrasound (US) as possible prognostic factors in heart failure. Their results are sound and easily transferable to clinical practice. However, this study also raises relevant technical concerns. First, the authors employed a 1.5–3.6 MHz phased array for both echocardiography and lung US. This probe, ideal for echocardiography, invariably generates a higher number of pleural artefacts due to its enhanced US reflection, which noticeably emphasizes the B line, namely the ring-down reverberation artefacts, that can be seen as continuous hyperechoic bands, parallel to the direction of the US beam, continuously moving along the pleural line with respiratory excursion, with respect to high-frequency probes. For instance, the thickness of the pleural line of the same subject would be 0.7–1.6 mm using a high-frequency probe, and 1.4–2.6 mm with a 3.5–5 MHz probe.2 This happens because the hyperechoic (‘pleural’) line does not correspond to any anatomical structure of normal lungs, but is generated by the remarkably different acoustic impedance between the chest wall and intrapulmonary air, also present in the pneumonectomy space.3 In addition, even the echocardiographic set-up of the probe (in terms of frequency, probe depth, time gain compensation, tissue harmonics, and electronically focused on this pleural line) could amplify B-line generation.4 Secondly, the patients were investigated only in the supine or near to supine position, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . which allows only the anterior and lateral chest to be scanned. This is not a trivial point, because chest US scan at best explores 70% of the pleural surface, and only about half of the latter surface is investigated in the supine position. Thus it is noteworthy that the basal-posterior lung zones, possibly collecting more fluid because of gravity and higher vascular space, remain unexplored. Besides this, the supine position does not allow identification of even the small fluid collection capable of enhancing B-lines or ‘ring down’. Finally, several co-morbidities besides those identified by the authors as exclusion criteria have been proved to increase the number of artefacts.5 These disorders could in turn account for the higher mortality and hospital readmission rate of patients with the highest artefact number.6 Other still unanswered questions concern the intraand interobserver repeatability of the measurements, which has been highly debated.7 Repeatable results could allow comparison of the lung fluid content before and after therapy, and testing of the predictive value of this change. Moreover, the clinical utility of chest US as a complementary tool in patients with heart failure would be better tested by investigating patients of the same NYHA class, receiving a comparable intensity of treatment. Conflict of interest: none declared.