Saverio Dragoni

Sildenafil Prevents Endothelial Dysfunction Induced by Ischemia and Reperfusion via Opening of Adenosine Triphosphate–Sensitive Potassium Channels A Human In Vivo Study

Background—Animal studies have demonstrated that administration of sildenafil can limit myocardial damage induced by prolonged ischemia, an effect that appears to be mediated by opening of adenosine triphosphate–sensitive potassium (KATP) channels. No study has investigated whether sildenafil can also prevent the impairment in endothelium-dependent vasodilatation induced by ischemia-reperfusion (IR) in humans. Methods and Results—In a double-blind, placebo-controlled, crossover design, 10 healthy male volunteers (25 to 45 years old) were randomized to oral sildenafil (50 mg) or placebo. Two hours later, endothelium-dependent, flow-mediated dilatation (FMD) of the radial artery was measured before and after IR (15 minutes of ischemia at the level of the brachial artery followed by 15 minutes of reperfusion). Seven days later, subjects received the other treatment (ie, placebo or sildenafil) and underwent the same protocol. Pre-IR radial artery diameter and FMD, as well as baseline radial artery diameter after IR, were similar between visits (P=NS). After placebo administration, IR significantly blunted FMD (before IR: 7.9±1.1%; after IR: 1.2±0.7%, P<0.01). Importantly, sildenafil limited this impairment in endothelium-dependent vasodilatation (before IR: 7.0±0.9%; after IR: 6.2±1.1%, P=NS; P<0.01 compared with placebo). In a separate protocol, this protective effect was completely prevented by previous administration of the sulfonylurea glibenclamide (glyburide, 5 mg), a blocker of KATP channels (n=7; FMD before IR: 10.3±1.5%; after IR: 1.3±1.4%, P<0.05). Conclusions—In humans, oral sildenafil induces potent protection against IR-induced endothelial dysfunction through opening of KATP channels. Further studies are needed to test the potential clinical implications of this finding.

Assessment of vascular function: flow-mediated constriction complements the information of flow-mediated dilatation

Objective: To determine whether vascular function assessed by low-flow-mediated constriction (L-FMC), a novel non-invasive method, complements the information obtained with “traditional” flow-mediated dilatation (FMD). Design and patients: In protocol 1, 12 healthy young volunteers underwent FMD and L-FMC measurements at rest and immediately after isometric exercise of the same hand. In protocol 2, 24 patients with coronary artery disease, 24 with congestive heart failure, 24 hypertensive patients and 64 healthy volunteers were enrolled to undergo L-FMC and FMD measurements. Results: In protocol 1, exercise was associated with mean (SD) increases in radial artery blood flow, diameter and L-FMC (from −5.1 (1.5)% to −7.8 (3.4)%, p<0.05), while FMD was significantly blunted (from 6.0 (2.4)% to 3.0 (3.2)%, p<0.05). In protocol 2, both FMD and L-FMC were blunted in the patient groups. Receiver operating curve analysis showed that, as compared with FMD alone, the combination of L-FMC and FMD significantly improved the sensitivity and specificity in detecting patients diagnosed with cardiovascular disease (p<0.05). Conclusion: In the first protocol, FMD and L-FMC were shown to be reciprocally regulated. A blunted FMD may, in certain cases, be an expression of increased resting vascular activation and not only of impaired endothelial function. In the second protocol, a statistical approach showed that implementation of L-FMC provides a better characterisation than FMD of vascular function in cardiovascular disease. Vascular (endothelial) function is a complex phenomenon which requires a multifaceted approach; it is suggested that a combination of L-FMC and FMD will provide additive and complementary information to “traditional” FMD measurements.

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.

Anti-Ro/SSA-Associated Corrected QT Interval Prolongation in Adults: The Role of Antibody Level and Specificity

Recent evidence suggests that anti‐Ro/SSA antibodies, strongly associated with the development of congenital heart block, may also be arrhythmogenic for the adult heart. In fact, anti‐Ro/SSA–positive patients with connective tissue disease (CTD) frequently display corrected QT (QTc) prolongation associated with an increase in ventricular arrhythmias. However, QTc prolongation prevalence markedly differs throughout the studies (10–60%), but the reason why is not yet clear. The aim of this study was to evaluate whether anti‐Ro/SSA–associated QTc prolongation in adult patients with CTD is related to antibody level and specificity.

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.

Model-based analysis of flow-mediated dilation and intima-media thickness

We present an end-to-end system for the automatic measurement of flow-mediated dilation (FMD) and intima-media thickness (IMT) for the assessment of the arterial function. The video sequences are acquired from a B-mode echographic scanner. A spline model (deformable template) is fitted to the data to detect the artery boundaries and track them all along the video sequence. The a priori knowledge about the image features and its content is exploited. Preprocessing is performed to improve both the visual quality of video frames for visual inspection and the performance of the segmentation algorithm without affecting the accuracy of the measurements. The system allows real-time processing as well as a high level of interactivity with the user. This is obtained by a graphical user interface (GUI) enabling the cardiologist to supervise the whole process and to eventually reset the contour extraction at any point in time. The system was validated and the accuracy, reproducibility, and repeatability of the measurements were assessed with extensive in vivo experiments. Jointly with the user friendliness, low cost, and robustness, this makes the system suitable for both research and daily clinical use.