Aversion to ambiguity and willingness to take risks affect therapeutic decisions in managing atrial fibrillation for stroke prevention: results of a pilot study in family physicians.

BACKGROUND: Anticoagulation is the therapeutic paradigm for stroke prevention in patients with atrial fibrillation (AF). It is unknown how physicians make treatment decisions in primary stroke prevention for patients with AF. OBJECTIVES: To evaluate the association between family physicians' risk preferences (aversion risk and ambiguity) and therapeutic recommendations (anticoagulation) in the management of AF for primary stroke prevention by applying concepts from behavioral economics. METHODS: Overall, 73 family physicians participated and completed the study. Our study comprised seven simulated case vignettes, three behavioral experiments, and two validated surveys. Behavioral experiments and surveys incorporated an economic framework to determine risk preferences and biases (e.g., ambiguity aversion, willingness to take risks). The primary outcome was making the correct decision of anticoagulation therapy. Secondary outcomes included medical errors in the management of AF for stroke prevention. RESULTS: Overall, 23.3% (17/73) of the family physicians elected not to escalate the therapy from antiplatelets to anticoagulation when recommended by best practice guidelines. A total of 67.1% of physicians selected the correct therapeutic options in two or more of the three simulated case vignettes. Multivariate analysis showed that aversion to ambiguity was associated with appropriate change to anticoagulation therapy in the management of AF (OR 5.48, 95% CI 1.08-27.85). Physicians' willingness to take individual risk in multiple domains was associated with lower errors (OR 0.16, 95% CI 0.03-0.86). CONCLUSION: Physicians' aversion to ambiguity and willingness to take risks are associated with appropriate therapeutic decisions in the management of AF for primary stroke prevention. Further large scale studies are needed.

Effects of estradiol on measurements of conduit artery endothelial function after ischemia and reperfusion in premenopausal women.

In premenopausal women, ovarian steroids are felt to play a role in the prevention of cardiovascular disease. We aimed to assess whether menstrual cycle variations in estrogen can modify the response to ischemia-reperfusion (IR) injury in humans. In an investigator-blinded crossover study, 10 healthy premenopausal women with regular menstrual cycles were studied. They had flow-mediated dilatation (FMD) measured by ultrasound in the radial artery before and after IR (15 min of brachial artery ischemia, 15 min of reperfusion) during both the early and late follicular phases of the menstrual cycle. The order of these visits was not randomized. IR significantly blunted FMD in the early follicular phase (pre-IR: 7.1% ± 1.0%; post-IR: 3.6% ± 1.0%, P = 0.01) when estradiol levels were low (148.4 ± 19.8 pmol/L). Conversely, FMD was preserved after IR during the late follicular phase (pre-IR: 7.2% ± 0.9%; post-IR: 7.0% ± 0.8%, P = NS, P = 0.03 compared with early follicular) when estradiol levels were high (825.7 ± 85.8 pmol/L, P < 0.001 compared with early follicular). There was a significant inverse relationship between estradiol concentration and IR-induced endothelial dysfunction (i.e., change in FMD after IR) (r = 0.59, r2 = 0.36, P < 0.01). These findings demonstrate, for the first time in humans, a clear relationship between the cyclical changes in serum concentrations of estradiol and the endothelium's response to IR.

Cyclooxygenase inhibition and rosuvastatin-induced vascular protection in the setting of ischemia-reperfusion: A human in vivo study.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitors have preconditioning effects involving up-regulation of cyclooxygenase (COX)-2. We investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against ischemia-reperfusion (IR)-induced endothelial dysfunction in the human forearm. Healthy volunteers (n=66) were allocated to placebo, acetylsalicylic acid (ASA) 81mg daily, ASA 325mg daily, celecoxib 200mg twice daily or 400mg ibuprofen four times daily, each administered for 5 to 7days. On the last day of study drug therapy, subjects received a single dose of 40mg rosuvastatin. Twenty-four hours later flow-mediated dilation (FMD) of the radial artery was evaluated before and after IR. In the placebo group, FMD was similar before and after IR (8.1±1.0 vs 7.2±0.8%; P=NS) indicating a significant protective effect of rosuvastatin. There was also no effect of IR on FMD in the ASA 81mg group (6.7±0.6 vs 6.1±0.7%; P=NS). In contrast, following IR there was a significant decrease in FMD in the ASA 325mg group (7.2±0.8 vs 3.3±0.7%, P<0.001), the celecoxib group (7.3±1.5 vs 2.6±1.5%, P<0.01) as well as the ibuprofen group (6.8±0.7 vs 2.6±0.8%; P<0.01). Therefore, nonselective COX inhibition with ASA 325mg and ibuprofen completely inhibit the protective effects of rosuvastatin in the setting of IR injury, as does therapy with the specific COX-2 antagonist celecoxib. In contrast, therapy with low dose ASA (81mg daily) does not have such inhibitory effects.

Repeated daily dosing with sildenafil provides sustained protection from endothelial dysfunction caused by ischemia and reperfusion: a human in vivo study.

Sildenafil and nitroglycerin (GTN) are effective pharmacological preconditioning agents, protecting from the adverse effects of ischemia and reperfusion (I/R). The objective of the present study was to determine whether repeated, daily administration of sildenafil or GTN provides sustained preconditioning from I/R in the human forearm vasculature. Thirty-six healthy volunteers participated in this investigator-blind, randomized, placebo-controlled trial. Subjects received transdermal GTN (0.6 mg/h, 2 h/day), sildenafil (50 mg once daily), or placebo. Twenty-four hours after the first dose of medication, subjects underwent an assessment of flow-mediated dilation (FMD) before and after I/R (15 min of upper arm ischemia followed by 15 min of reperfusion). Subjects continued their study medication for 7 days, at which point FMD measurements were repeated before and after I/R. Venous blood samples were obtained for the determination of myeloperoxidase, P-selectin, and myoglobin before and after each I/R episode. Twenty-four hours after the first dose, both sildenafil and GTN (but not placebo) provided protection from the adverse effects of I/R. After 7 days of repeated daily doses and 24 h after the last dose, FMD was significantly blunted after I/R in placebo- and GTN-treated groups. In contrast, repeated daily administration of sildenafil provided continued protection from the adverse effects of I/R on endothelial function. There was no significant change in plasma levels of myeloperoxidase, P-selectin, or myoglobin at any time point. In conclusion, the present study establishes, for the first time in humans, that sildenafil, but not GTN, provides sustained pharmacological preconditioning of the endothelium and protection from adverse I/R effects on vascular function.

Daily ischemic preconditioning provides sustained protection from ischemia-reperfusion induced endothelial dysfunction: a human study.

BACKGROUND: It is well established that acute ischemic preconditioning (IPC) protects against ischemia-reperfusion (IR) injury; however, the effectiveness of repeated IPC exposure has not been extensively investigated. We aimed to determine whether daily IPC episodes provide continued protection from IR injury in a human forearm model, and the role of cyclooxygenase-2 in these responses. METHODS AND RESULTS: Thirty healthy volunteers were randomized to participate in 2 of 3 protocols (IR alone, 1-day IPC, 7-day IPC) in an operator-blinded, crossover design. Subjects in the IR alone protocol underwent flow-mediated dilation (FMD) measurements pre- and post-IR (15' upper-arm ischemia and 15' reperfusion). The 1-day IPC protocol involved FMD measurements before and after 1 episode of IPC (3 cycles of 5' upper-arm ischemia and 5' reperfusion) and IR. Day 7 of the 7-day IPC protocol was identical to the 1-day IPC protocol but was preceded by single daily episodes of IPC for 6 days prior. During each protocol, subjects received a 7-day treatment of either the cyclooxygenase-2 inhibitor celecoxib or placebo. Pre-IR FMD was similar between groups. IR alone reduced FMD post-IR (placebo, ΔFMD: -4.4±0.7%; celecoxib, ΔFMD: -5.0±0.5%). One-day IPC completely prevented this effect (placebo, ΔFMD: -1.1±0.6%; celecoxib, ΔFMD: 0.0±0.7%; P<0.0001). Similarly, 7-day IPC demonstrated persistent endothelial protection post-IR (placebo, ΔFMD: -0.9±0.9%; celecoxib, ΔFMD: 0.0±0.8%; P<0.0001, P<0.0001 for ANOVA effect of IPC protocol). Celecoxib did not alter responses to IR in any protocol. CONCLUSIONS: Daily episodes of IPC provide sustained protection from IR-induced endothelial dysfunction in humans through a mechanism that appears cyclooxygenase-2-independent.

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.

Inhibition of sPLA2 and endothelial function: a substudy of the SPIDER-PCI trial.

BACKGROUND: Inflammation plays an important role in the pathophysiology of atherosclerosis and endothelial dysfunction, and occurs after percutaneous coronary intervention (PCI). We evaluated whether endothelial function is attenuated after PCI and if inhibition of secretory phospholipase A2 (sPLA2) activity augments endothelial function and coronary flow reserve (CFR) in these patients. METHODS: In the sPLA2 Inhibition to Decrease Enzyme Release After Percutaneous Coronary Intervention (SPIDER-PCI) study, patients undergoing elective PCI were randomized to receive Varespladib (Anthera Pharmaceuticals Inc, San Mateo, CA), an inhibitor of sPLA2, or placebo 3-5 days prior to PCI and for 5 days after PCI. In this substudy, endothelial function was assessed in 31 patients by flow-mediated dilation (FMD) before treatment and on the day after PCI, while taking study medication. During the PCI procedure, CFR was assessed using a Doppler guide wire. RESULTS: Baseline and procedural characteristics were comparable in both groups and sPLA2 activity was similar at baseline. After PCI, sPLA2 activity decreased only in the Varespladib group (2.9 ± 0.9 to 0.5 ± 0.4 ng/mL), and high-sensitivity C-reactive protein (hsCRP) increased by more than 100% in both groups. FMD at baseline was 3.66 ± 2.45% (Varespladib) and 3.37 ± 1.73% (placebo) with nonsignificant increase in both groups after PCI. The effect of Varespladib on FMD, adjusted for pre-PCI FMD by linear regression, was -1.16 ± 1.68%; P = 0.5. CFR was 2.45 ± 0.66 and 2.77 ± 0.85 in the Varespladib and placebo groups, respectively (P = 0.36). CONCLUSIONS: Systemic endothelial function is not reduced after elective PCI despite eliciting acute inflammatory response. Acute inhibition of sPLA2 activity with Varespladib does not affect endothelial or microvascular function after PCI.

Loss of the preconditioning effect of rosuvastatin during sustained therapy: a human in vivo study.

Studies have demonstrated that the acute administration of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has protective effects in the setting of ischemia-reperfusion (IR). Previously, we demonstrated that a single dose of rosuvastatin prevented IR-induced endothelial dysfunction in humans through a cyclooxygenase-2-dependent mechanism. Whether the chronic administration of HMG-CoA reductase inhibitors provides similar protection remains controversial and is unknown in humans. Eighteen male volunteers were randomized to receive a single dose of rosuvastatin (20 mg) or placebo. Twenty-four hours later, endothelium-dependent, radial artery flow-mediated dilation (FMD) was measured before and after IR (15 min of upper arm ischemia followed by 15 min of reperfusion). In a separate protocol, 30 healthy volunteers were randomized in a double-blind fashion to receive oral rosuvastatin (20 mg/day) and placebo, rosuvastatin, and celecoxib (100 mg bid) or placebo alone, all for 21 days. Twenty-four hours after the final administration of study medication, FMD was measured before and after IR. Pre-IR FMD was similar between groups in both protocols. In the acute administration protocol, rosuvastatin significantly prevented the blunting of FMD associated with IR (FMD pre-IR: 8.4 ± 1.3%; post-IR: 6.2 ± 1.3%; P = 0.01 ANOVA, treatment group interaction). In the daily administration protocol, IR significantly blunted FMD in the placebo group (FMD pre-IR: 7.5 ± 0.9%; post-IR: 3.3 ± 0.7%; P < 0.001). Chronic treatment with rosuvastatin did not modify this ischemic injury (FMD pre-IR: 6.9 ± 0.4%; post-IR: 1.6 ± 1.0%; P < 0.001; P = NS ANOVA, treatment group interaction). Similarly, FMD responses post-IR in volunteers receiving rosuvastatin and celecoxib did not significantly differ from placebo (FMD pre-IR: 8.3 ± 0.9%; post-IR: 2.1 ± 0.8%; P < 0.001; P = NS ANOVA, treatment group interaction). In contrast to acute administration, chronic rosuvastatin does not prevent the development of IR-induced endothelial dysfunction in normal humans.

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.

Chronic pharmacological preconditioning against ischemia.

Despite decades of research and thousands of experimental publications, acute preconditioning strategies have yet to be implemented in clinical practice. While some have attributed this to a failure of the experimental studies to mimic the clinical environment, others have suggested that acute preconditioning strategies themselves may possess physiological limitations. In particular, there is evidence to suggest a reduced efficacy of acute preconditioning in the aged heart and in disease states, such as diabetes, hypertension, hyperlipidemia, and atherosclerosis. In addition, pharmacologic agent commonly used in clinical practice, such as sulfonylureas and non-steroidal anti-inflammatory agents may interfere with acute preconditioning signaling pathways. Such considerations may preclude the translation of acute preconditioning strategies to the clinical setting. This has led some to shift attention to alternate strategies of cardioprotection, one such strategy being the possibility of generating a prolonged state of cardioprotection. Although preliminary, studies to date have suggested that sustained preconditioning strategies may not be associated with the same drawbacks as acute preconditioning. Further, cardioprotective signaling pathways that elicit the sustained preconditioning response may be distinct from acute signaling pathways, which permit pharmacologic targeting of these pathways in the future. Additionally, sustained preconditioning strategies may be clinically applicable in the setting of acute myocardial infarction, a setting where acute preconditioning strategies are inherently limited. This review will briefly discuss the current data regarding sustained preconditioning strategies, including those in humans, and discuss the goal of future studies in this setting.

The acute administration of either amiloride or captopril does not prevent endothelial dysfunction induced by ischemia and reperfusion in the human forearm vasculature.

Animal studies have demonstrated the ability of both sodium-hydrogen exchange inhibitors and angiotensin-converting enzyme inhibitors to reduce infarct size and preserve postischemic ventricular function following ischemia and reperfusion (IR) injury. Whether these interventions can also prevent IR-induced impairment of endothelial function in humans has not been investigated. We performed 2 separate double-blind, placebo-controlled, crossover studies. In the first study, 10 healthy volunteers were randomized to receive oral amiloride (10 mg) or a placebo. In a separate study, another group of volunteers (n = 10) was randomized to receive oral captopril (50 mg) or a placebo. At the time of the peak hemodynamic effect of the drug (3 and 1.5 h after administration of amiloride and captopril, respectively), endothelium-dependent, flow-mediated dilatation of the radial artery was measured before and after IR. IR significantly blunted flow-mediated dilatation in all groups (placebo: pre-IR: 6.8% ± 0.7%; post-IR: 2.9% ± 0.9%; P < 0.01; amiloride: pre-IR: 5.9% ± 0.6%; post-IR: 2.1% ± 1.3%; P = 0.01; captopril: pre-IR: 6.0% ± 0.5%; post-IR: 2.0% ± 0.6%; P < 0.01). In humans, neither 10 mg of oral amiloride nor 50 mg of oral captopril was able to provide protection against IR-induced endothelial dysfunction in the peripheral vasculature.

The endothelial-protective effects of HMG-CoA reductase inhibition in the setting of ischemia and reperfusion injury.

Animal studies have consistently demonstrated the ability of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors to limit the damage induced by ischemia-reperfusion (IR) in the cardiac, cerebral and mesenteric circulation through a mechanism dependent on the upregulation of cyclooxygenase-2 (COX-2). Our group performed studies aimed at investigating the mechanism of HMG-CoA reductase inhibitor-mediated endothelial protection from IR injury, in particular the role of COX-2, in a human in vivo model of IR-induced endothelial dysfunction. We demonstrated that HMG-CoA reductase inhibition protects against IR-induced endothelial damage, an effect that was lost upon COX-2 inhibition. These observations may suggest a mechanistic explanation for the cardioprotection observed in clinical settings such as percutaneous coronary interventions and coronary artery bypass surgery and may also propose a mechanistic hypothesis for the reported cardiotoxic effects of cyclooxygenase-2 inhibitors observed in clinical studies. These studies are summarized and discussed in the present paper.

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.

Rosuvastatin prevents conduit artery endothelial dysfunction induced by ischemia and reperfusion by a cyclooxygenase-2-dependent mechanism.

OBJECTIVES: The purpose of this study was to determine whether single-dose rosuvastatin (40 mg) protects against ischemia and reperfusion (IR)-induced endothelial dysfunction in humans and whether this effect is cyclooxygenase (COX)-2 dependent. BACKGROUND: Animal studies have demonstrated that rosuvastatin can limit damage and improve recovery after IR. METHODS: In a double-blind, parallel design, 20 volunteers were randomized to a single dose of oral rosuvastatin (40 mg) or placebo. Twenty-four hours later, endothelium-dependent, flow-mediated dilation (FMD) of the radial artery was measured before and after IR (15 min of upper arm ischemia followed by 15 min of reperfusion). In a separate protocol, 18 volunteers received the COX-2 inhibitor celecoxib (200 mg orally twice daily) for 5 days. On day 4, subjects were randomized to single-dose rosuvastatin (40 mg) or placebo and 24 h later underwent the same protocol as described. RESULTS: Pre-IR FMD was similar between groups (p = NS). IR significantly blunted FMD in the placebo group (FMD pre-IR: 6.4 +/- 1.4%; FMD post-IR: 1.1 +/- 3.8%, [p = 0.002]). Rosuvastatin prevented this impairment (FMD pre-IR: 7.5 +/- 3.1%; FMD post-IR: 6.2 +/- 3.9%, [p = NS] vs. rosuvastatin pre-IR, [p = 0.03] vs. placebo). Pre-treatment with celecoxib completely abolished rosuvastatin's protective effect (FMD pre-IR: 8.0 +/- 2.2%; FMD post-IR: 1.4 +/- 2.0%, [p < 0.001] compared with pre-IR, [p = NS] vs. placebo, [p = 0.002] vs. rosuvastatin alone). CONCLUSIONS: Rosuvastatin pharmacologically prevents the development of IR-induced conduit artery endothelial dysfunction. This beneficial effect of rosuvastatin is mediated by a COX-2-dependent mechanism, evidence that may also provide potential mechanistic insight into the reported cardiotoxic effects of COX-2 inhibitors.

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.