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.

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.

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.

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.

Endothelium and haemorheology.

The vascular endothelium has been recognized to have a central importance in maintaining vascular homeostasis and in preventing cardiovascular disease. The mechanisms underlying the regulation of its function are extremely complex, and are principally determined by physical forces imposed on the endothelium by the flowing blood. In the present paper, we describe the interactions between the rheological properties of blood and the vascular endothelium. The role of shear stress, viscosity, cell-cell interactions, as well as the molecular mechanisms that are important for the transduction of these signals are discussed both in physiology and in pathology, with a particular attention to the role of reactive oxygen species. In the final conclusions, we propose an hypothesis regarding the implications of changes in blood viscosity, and particularly on the significance of secondary hyperviscosity syndromes.

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 (K(ATP)) 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 K(ATP) 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 K(ATP) channels. Further studies are needed to test the potential clinical implications of this finding.

The effect of ischemia and reperfusion on microvascular function: a human in vivo comparative study with conduit arteries.

Although microvascular dysfunction is of critical importance in the pathophysiology of myocardial ischemic syndromes, no study has investigated whether there are differences in the sensitivity to ischemia and reperfusion injury between microvessels and conduit arteries. Ten healthy young nonsmoking male volunteers (age range 24-45) were enrolled. Parameters measured included radial (conduit) artery (endothelium-dependent) flow-mediated dilation, microvascular cutaneous reactive hyperemia (using laser Doppler) and acetylcholine-induced microvascular vasodilation (laser Doppler iontophoresis). Data were acquired before and after ischemic injury (15 minutes of ischemia of the brachial artery followed by 15 minutes reperfusion) and analyzed in a randomized, blinded fashion. Conduit artery FMD was significantly blunted after ischemia (before: 7.5 +/- 1.1%; after: 2.9 +/- 1.0%, P < 0.05). Conversely, ischemia had no effect on microvascular reactive hyperemia (P = ns) and acetylcholine-induced vasodilation (P = ns). Using a human in vivo model, we demonstrate that microvessels are more resistant to ischemic injury as compared to conduit arteries.

Correlation analysis between different parameters of conduit artery and microvascular vasodilation.

Impaired endothelial responsiveness to specific vasodilator stimuli has been used as a surrogate marker of cardiovascular risk. Multiple methods allow testing endothelial responses in both microvessels and conduit arteries, but it is still unclear whether there is a relationship in endothelial function between these two different vascular beds. Twenty-five healthy young non smoking male volunteers (age range 24-45) were enrolled. Radial (conduit) artery (endothelium-dependent) flow-mediated dilation (FMD), microvascular cutaneous reactive hyperemia (using laser Doppler) and acetylcholine-induced microvascular vasodilation (laser Doppler iontophoresis) were measured. Data were analyzed in a randomized fashion in order to test the existence of a correlation among these measures of endothelium (in)dependent vasodilation. Conduit artery FMD showed a negative correlation with resting radial artery diameter (R=0.44, P<0.05). There was a correlation between peak responsiveness to acetylcholine and peak reactive hyperemia (R=0.41, P<0.05). Conversely, absolutely no correlation was shown between FMD and measures of microvascular vasomotion, including reactive hyperemia (P=ns) and acetylcholine-induced vasodilation (P=ns). Using three different human in vivo models, we test conduit artery and microvascular endothelial vasodilation. While microvascular flow reserve measurements induced by endothelium-dependent and independent stimuli appear to be linearly correlated, we show no correlation in endothelium-dependent vasomotion between the micro- and macrocirculation.

Chronic protection against ischemia and reperfusion-induced endothelial dysfunction during therapy with different organic nitrates.

INTRODUCTION: Ischemic and pharmacologic preconditioning have great clinical potential, but it remains unclear whether their effects can be maintained over time during repeated exposure.We have previously demonstrated that the acute protective effect of nitroglycerin (GTN) is attenuated during repeated daily administration. Pentaerythrityl tetranitrate (PETN) is an organic nitrate with different hemodynamic and biochemical properties. The purpose of the current experiment was to study the preconditioning-like effects of PETN and GTN during repeated daily exposure. METHODS AND RESULTS: In a randomized, investigator-blind parallel trial, 30 healthy (age 25-32) volunteers were randomized to receive (1) transdermal GTN (0.6 mg/h) administered for 2 h a day for 6 days; (2) oral PETN (80 mg) once a day for 6 days; or (3) no therapy. One week later, endothelium-dependent flow-mediated dilation was assessed before and after exposure to ischemia and reperfusion (IR). IR caused a significant blunting of the endothelium-dependent relaxation in the control group (FMD before IR: 5.8 ± 2.1%; after IR 1.0 ± 2.1%; P < 0.01). Daily, 2-h exposure to GTN partially prevented IR-induced endothelial dysfunction (FMD before IR: 7.7 ± 2.4%; after IR 4.3 ± 3.0%; P < 0.01 compared to before IR). In contrast, daily PETN administration afforded greater protection from IR-induced endothelial injury (FMD before IR: 7.9 ± 1.7%; after IR 6.4 ± 5.3%, P = ns; P < 0.05 ANOVA across groups). In vitro, incubation of human endothelial cells with GTN (but not PETN) was associated with inhibition (P < 0.01) of aldehyde dehydrogenase, an enzyme that is important for both nitrate biotransformation and ischemic preconditioning. DISCUSSION: We previously showed that upon repeated administration, the preconditioning-like effects of GTN are attenuated. The present data demonstrate a gradient in the extent of protection afforded by the two nitrates, suggesting that PETN-induced preconditioning is maintained after prolonged administration in a human in vivo model of endothelial dysfunction induced by ischemia. Using isolated human endothelial cells, we propose a mechanistic explanation for this observation based on differential effects of GTN versus PETN on the activity of mitochondrial aldehyde dehydrogenase.

Anti-Ro/SSA-associated corrected QT interval prolongation in adults: the role of antibody level and specificity.

OBJECTIVE: 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. METHODS: Forty-nine adult patients with CTD underwent a resting 12-lead electrocardiogram recording to measure QTc interval, and a venous withdrawal to determine anti-Ro/SSA antibody level and specificity (anti-Ro/SSA 52 kd and anti-Ro/SSA 60 kd) by immunoenzymatic methods and Western blotting. RESULTS: In our population, a direct correlation was demonstrated between anti-Ro/SSA 52-kd level and QTc duration (r = 0.38, P = 0.007), patients with a prolonged QTc had higher levels of anti-Ro/SSA 52 kd with respect to those with a normal QTc (P = 0.003), and patients with a moderate to high level (≥50 units/ml) of anti-Ro/SSA 52 kd showed a longer QTc interval (P = 0.008) and a higher QTc prolongation prevalence (P = 0.008) than those with a low positive/negative level (<50 units/ml). On the contrary, no association was found between QTc and anti-Ro/SSA 60-kd level. CONCLUSION: In anti-Ro/SSA-positive adult patients with CTD, the occurrence of QTc prolongation seems strictly dependent on the anti-Ro/SSA 52-kd level. This finding, possibly explaining the different QTc prolongation prevalence reported, strengthens the hypothesis that an extremely specific autoimmune cross-reaction is responsible for the anti-Ro/SSA-dependent interference on ventricular repolarization.

Conduit artery constriction mediated by low flow a novel noninvasive method for the assessment of vascular function.

OBJECTIVES: We describe and validate a novel noninvasive method that complements the data from "traditional" flow-mediated dilation (FMD) studies. BACKGROUND: The study of peripheral vascular reactivity provides important diagnostic and prognostic information in patients with (or at risk for) cardiovascular disease. METHODS: High-resolution ultrasound and automatic computerized analysis were used to measure the diameter of the radial artery at rest and in conditions of locally decreased and increased shear stress (respectively, low-flow-mediated constriction [L-FMC] and flow-mediated dilation [FMD]). A composite end point (L-FMC + FMD) was also calculated. A total of 196 studies were performed. RESULTS: When the repeatability of the method was tested, the range of variation across measurements was 1.1% for L-FMC and 1.7% for FMD; the intraclass correlation coefficient was 0.80 and 0.68, respectively. Low-flow-mediated constriction, FMD, and their composite end point were significantly blunted after acute smoking, in coronary artery disease patients, and in hypertensive patients as compared with that seen in healthy age-matched volunteers (p < 0.01, analysis of variance). Low-flow-mediated constriction, but not FMD, was blunted (p < 0.05) after administration of fluconazole (an inhibitor of a cytochrome P450-derived endothelium-derived hyperpolarization factor) and aspirin (an inhibitor of cyclooxygenase). Flow-mediated dilation, but not L-FMC, was blunted (p < 0.05) by nitric oxide synthase inhibition. CONCLUSIONS: Low-flow-mediated constriction is a simple, rapid, and accurate measure of resting arterial tone that does not require further procedures as compared with "traditional" FMD measurements. While FMD measures endothelial responses to sudden increases in shear stress, L-FMC is a measure of the response to resting shear stress levels, and, therefore, it provides additional information that is complementary to FMD.

Nitroglycerin protects the endothelium from ischaemia and reperfusion: human mechanistic insight.

AIMS: Nitroglycerin (GTN) modulates tissue damage induced by ischaemia and reperfusion (IR) in a mechanism that is similar to ischaemic preconditioning. We set out to study, using a human model of endothelial IR injury, whether GTN-induced endothelial preconditioning is mediated by reactive oxygen species (ROS) formation and/or opening of mitochondrial permeability transition pores (mPTP). METHODS: In two double-blind, randomized, parallel studies, a total of 66 volunteers underwent measurement of radial artery endothelium-dependent, flow-mediated dilation (FMD) before and after local IR. Subjects were treated, 24 h before IR, with different drugs in order to test the mechanism of GTN-induced endothelial protection. RESULTS: Transdermal GTN (0.6 mg h(-1) for 2 h, administered 24 h before IR) significantly reduced the impairment of FMD caused by IR (placebo group: FMD after IR, 1.3 +/- 0.8%; GTN group: FMD after IR, 5.3 +/- 0.9%, P < 0.01 compared with placebo). This protective effect was lost when vitamin C (2 g i.v. at the time of GTN administration) or ciclosporin (an inhibitor of mPTP, 100 mg 2 h prior to GTN administration) were coadministered (FMD after IR: vit C + GTN group, 2.1 +/- 1.0%; ciclosporin + GTN group, 1.7 +/- 0.8%; both P < 0.05 compared with GTN alone). CONCLUSIONS: We demonstrate that GTN protects the endothelium against IR-induced endothelial dysfunction, in an effect similar to delayed ischaemic preconditioning. Using a human model, we provide evidence supporting the concept that this protective effect is mediated by ROS release and mPTP opening upon GTN administration.

Postconditioning fails to prevent radial artery endothelial dysfunction induced by ischemia and reperfusion: evidence from a human in vivo study.

Animal studies have shown that, as compared with unrestricted reperfusion, exposure to brief periods of controlled ischemia (postconditioning) at the end of a prolonged ischemia reduces the extent of tissue damage. We set out to test whether postconditioning can prevent endothelial dysfunction induced by ischemia and reperfusion in a human in vivo model. Ten healthy young non-smoking volunteers were enrolled in this cross-over, controlled, investigator-blinded study. Subjects were exposed to 15 min of forearm ischemia followed by either unrestricted reperfusion or postconditioning (3 periods of 20 s of ischemia separated by 10 s of reperfusion). Endothelium-dependent flow-mediated dilation (FMD) was measured at the level of the radial artery before and after ischemia (with or without postconditioning). Forearm ischemia blunted FMD in both study visits (unrestricted reperfusion visit: before ischemia, 7.7% +/- 1.3%; after ischemia, 2.5% +/- 1.4%; and postconditioning visit: before, 7.3% +/- 1.2%; after, 2.6 +/- 1.6%; P < 0.05 for both, P = not significant (NS) between visits). In contrast with data from animal studies, postconditioning (20 s ischemia-- 10 s reperfusion repeated 3 times) does not limit post-ischemic endothelial dysfunction in this human in vivo model. Further human studies are necessary to evaluate other reperfusion protocols in an attempt to limit post-ischemic tissue damage.

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.