Postconditioning Protects Against Endothelial Ischemia-Reperfusion Inj
http://www.100md.com
《循环学杂志》
the Vascular Physiology Unit (S.P.L., A.T.P., J.E.D.), Institute of Child Health
Hatter Cardiovascular Institute and Centre for Cardiology (D.M.Y.)
Centre for Clinical Pharmacology and Therapeutics (S.P.L., A.T.P., R.J.M.), University College London, London, United Kingdom.
Abstract
Background— Hypoxic cell death follows interruption of blood supply to tissues. Although successful restoration of blood flow is mandatory for salvage of ischemic tissues, reperfusion can paradoxically place tissues at risk of further injury. Brief periods of ischemia applied at the onset of reperfusion have been shown to reduce ischemia-reperfusion (IR) injury, a phenomenon called postconditioning. The aim of this study was to determine whether postconditioning protects against endothelial IR injury in humans, in vivo.
Methods and Results— Brachial artery endothelial function was assessed by vascular ultrasound to measure flow-mediated dilation (FMD) in response to forearm reactive hyperemia. FMD was measured before and after IR (20 minutes of arm ischemia followed by 20 minutes of reperfusion) in healthy volunteers. To test the protective effects of postconditioning, 3 cycles of reperfusion followed by ischemia (each lasting 10 or 30 seconds) were applied immediately after 20 minutes of arm ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before the application of the 10-second postconditioning stimulus. IR caused endothelial dysfunction (FMD 9.1±1.2% pre-IR, 3.6±0.7% post-IR, P<0.001; n=11), which was prevented by postconditioning applied as 10-second cycles of reperfusion/ischemia (FMD 9.9±1.7% pre-IR, 8.3±1.4% post-IR, P=NS; n=11) and 30-second cycles of reperfusion/ischemia (FMD 10.8±1.7% pre-IR, 9.5±1.5% post-IR, P=NS; n=10) immediately at the onset of reperfusion. No protection was observed when the application of the 10-second postconditioning stimulus was delayed for 1 minute after the onset of reperfusion (FMD 9.8±1.2% pre-IR, 4.0±0.9% post-IR, P<0.001; n=8).
Conclusions— This study demonstrates for the first time that postconditioning can protect against endothelial IR injury in humans. Postconditioning might reduce tissue injury when applied at the onset of reperfusion by modifying the reperfusion phase of IR.
Key Words: endothelium ischemia reperfusion reperfusion injury postconditioning
Introduction
The prerequisite for salvage of viable myocardium and limitation of infarct size after an acute ischemic event is timely reperfusion.1,2 However, restoration of coronary blood flow carries with it the risk of further myocardial injury (reperfusion injury).3 Events occurring during reperfusion have been shown to be responsible in part for reversible (stunning) and, more important, lethal (necrosis/apoptosis) injury in animal models.4–7 Although the contribution of reperfusion injury to infarct size in humans is unknown, it is possible that minimizing reperfusion injury might reduce infarct size in humans.
Clinical Perspective p 1019
Modulating blood flow on reperfusion (through gradual or intermittent reperfusion) has been shown to reduce experimental infarct size.8,9 In particular, Vinten-Johansen et al10 have shown that a schedule of intermittent reperfusion (termed "postconditioning") applied at the onset of reperfusion reduces infarct size in animal models of myocardial ischemia-reperfusion (IR) injury. The protective effect of postconditioning is of a similar magnitude to that seen with ischemic preconditioning.11 However, unlike preconditioning, the influence of postconditioning is restricted to the reperfusion phase of IR injury, and this has renewed interest in the reperfusion phase as a target for cardioprotection.12
Staat et al13 have recently shown that a postconditioning protocol of intermittent reperfusion reduces myocardial injury during primary angioplasty in patients with acute myocardial infarction. We have developed a model of IR injury that results in endothelial dysfunction in conduit14 and resistance vessels.15 In the present study, we used this model to determine the potential protective effects of different schedules of ischemic postconditioning against endothelial IR injury in humans, in vivo.
Methods
Subjects
Forty studies were performed on 11 healthy volunteers (6 men, 5 women; mean age±SD 23.6±4.2 years; range 18 to 33 years). All volunteers gave informed consent. Studies were approved by the local research ethics committee and performed in a temperature-controlled laboratory (24°C to 26°C). All studies repeated in the same volunteers were at least 7 days apart.
Induction of IR
The nondominant arm was made ischemic by the inflation of a 9-cm-wide blood pressure cuff placed around the upper part of the arm to a pressure of 200 mm Hg for 20 minutes, as described previously.14,16
Induction of Postconditioning
Postconditioning was induced by the application of intermittent short periods of ischemia and reperfusion on the nondominant arm early during reperfusion. After index ischemia, the arm was allowed to reperfuse for 10 seconds, after which the blood pressure cuff was inflated again to 200 mm Hg, which made the arm ischemic for 10 seconds. This deflation/inflation cycle was repeated a total of 3 times (1 minute total duration; PostC10). The effect of longer cycles of postconditioning (3 cycles of 30 seconds of reperfusion alternating with 30 seconds of ischemia) was also determined (PostC30).
Assessment of Conduit Vessel Endothelial Function
Endothelial function of the brachial artery was assessed by flow-mediated dilation (FMD) of the brachial artery in the nondominant arm, as described previously.17 In this technique, reactive hyperemia of the forearm is used to increase blood flow in the brachial artery, resulting in brachial artery dilatation. A B-mode scan of the brachial artery was obtained in longitudinal section between 5 and 10 cm above the antecubital fossa with a 7.0-MHz linear-array transducer (spatial resolution of 0.1 mm18) and a standard Acuson XP10 system. Longitudinal, ECG-gated, end-diastolic images were acquired every 3 seconds for offline analysis. Arterial diameter over a 1- to 2-cm segment was determined for each image with the use of automatic edge-detection software (Brachial Tools, Medical Imaging Applications). Blood flow was manipulated in the brachial artery by a 7-cm-wide blood pressure cuff placed around the forearm immediately below the antecubital fossa. After 1 minute of baseline flow, the cuff was inflated to 300 mm Hg for 5 minutes and released, which resulted in a brief episode of reactive hyperemia. Brachial artery diameter changes in response to blood flow were assessed for an additional 5 minutes. Blood flow velocity was monitored continuously by pulsed-wave Doppler.
Experimental Protocols
Effect of IR on Vascular Dilator Function
To determine the effect of IR on endothelial function, FMD was assessed before ischemia (baseline) and at 20 minutes after reperfusion (n=11; Figure 1, protocol a). We have previously demonstrated that this protocol results in brachial artery endothelial dysfunction but does not have an effect on vascular smooth muscle function, because it did not alter the dilator response to nitroglycerin (NTG).14
Effect of Postconditioning on Endothelial IR Injury
To establish that protection against endothelial IR can be achieved by modifying reperfusion, postconditioning was induced in the same group of volunteers. Three cycles of 10 seconds of reperfusion and 10 seconds of ischemia (PostC10; n=11; Figure 1, protocol b) or 3 cycles of 30 seconds of reperfusion and 30 seconds of ischemia (PostC30; n=10; Figure 1 protocol c) were applied immediately after index ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before application of the 10-second-cycle postconditioning stimulus (DelayedPostC10; n=8; Figure 1, protocol d).
Calculations and Statistics
All data are expressed as mean±SE unless otherwise stated. Brachial artery diameter was measured in millimeters and dilation expressed as percentage increase from baseline diameter. The FMD flow stimulus during reactive hyperemia was expressed as the ratio of peak to baseline volume flow per minute. Data were compared by paired Student t test or repeated-measures ANOVA as appropriate. For multiple comparisons (8 groups), probability values by ANOVA were Bonferroni adjusted. In all cases, P<0.05 was considered statistically significant.
Results
All subjects tolerated the procedures without any complications. There were no differences in the responses between men and women.
Effect of IR on Vascular Dilator Function
The IR protocol had no effect on blood pressure and heart rate (Table). IR reduced brachial artery FMD (9.1±1.2% pre-IR versus 3.6±0.7% post-IR, P<0.001, ANOVA; n=11; Figure 2a), which is consistent with endothelial dysfunction. These results could not be explained by differences in brachial artery diameter (3.4±0.2 mm pre-IR versus 3.4±0.2 mm post-IR, P=NS, t test) and FMD flow stimulus (peak to baseline volume flow ratio 11.3±1.5 pre-IR versus 10.9±1.0 post-IR, P=NS, t test; Table).
Summary of Pre- and Post-IR Data From Sequential Studies in Healthy Volunteers
Effect of Postconditioning on Endothelial IR Injury
Postconditioning had no effect on blood pressure, heart rate, arterial diameter, or flow stimulus during reactive hyperemia (Table). Ten-second-cycle postconditioning (PostC10), applied immediately at the onset of reperfusion, prevented IR-induced endothelial dysfunction (FMD 9.9±1.7% pre- versus 8.3±1.4% post-IR+PostC10, P=NS, ANOVA; n=11; Figure 2b). Similarly, 30-second-cycle postconditioning (PostC30) was also protective (FMD 10.8±1.7% pre- versus 9.5±1.5% post-IR+PostC30, P=NS, ANOVA; n=10; Figure 2c). However, no protection was observed when the application of the PostC10 stimulus was delayed for 1 minute after blood flow restoration to the arm (FMD 9.8±1.2% pre- versus 4.0±0.9% post-IR+DelayedPostC10, P<0.001, ANOVA; n=8; Figure 2d). Mean post-IR FMD in PostC10 and PostC30 studies was significantly higher than the respective values obtained in control (IR alone; P<0.001, ANOVA) and DelayedPostC10 experiments (P<0.001, ANOVA; Table).
Discussion
This study demonstrates for the first time in humans in vivo that ischemic postconditioning prevents endothelial IR injury in conduit vessels. The first moments of reperfusion are critical for endothelial protection by postconditioning, because the protective effect disappeared when the stimulus was applied 1 minute after the onset of reperfusion.
Ischemic postconditioning describes protection against IR injury by a series of brief interruptions of blood flow applied at the onset of reperfusion. Studies by Zhao et al11 and Halkos et al19 demonstrated in a canine model of myocardial infarction that the application of a postconditioning protocol immediately after coronary artery occlusion reduced myocardial infarct size, an effect comparable to that of ischemic preconditioning. Since then, the cardioprotective effects of postconditioning have been confirmed in the in vivo and ex vivo heart in rats20,21 and rabbits,22,23 as well as in isolated rat cardiomyocytes.24 More recently, Staat et al13 have demonstrated that postconditioning reduces infarct size in patients undergoing primary angioplasty for acute myocardial infarction.
In the present study, we used arm ischemia to model IR injury in humans. Previously, we have shown that ischemia causes transient endothelial dysfunction of the brachial artery, with preservation of smooth muscle function.14 This model is a convenient way to compare different schedules of postconditioning stimuli. In our IR model, the degree of protection by postconditioning is similar to that achieved by ischemic preconditioning and remote ischemic preconditioning in previous studies in healthy volunteers.14,16 This is in agreement with animal data19,21 and suggests that injury suffered during reperfusion is largely responsible for the IR-induced endothelial dysfunction we observed. Moreover, our present findings demonstrate that postconditioning induces protection against endothelial IR injury by modifying events that occur during the early stages of reperfusion20; a delay in the application of the postconditioning stimulus for as little as 1 minute resulted in loss of protection. Cycle length (10 versus 30 seconds) mattered less than the timing of the initiation of the postconditioning schedule. In this regard, it is worth noting that the recent study of primary angioplasty in patients used a postconditioning protocol of four 60-second cycles of ischemia/reperfusion, also initiated within the first minute of reperfusion.13 It remains to be determined to what extent these results in the brachial vasculature can be extrapolated to the coronary vasculature. Nonetheless, the present study provides corroborative evidence that postconditioning occurs in humans and has similar characteristics to those described in animal models and patients with acute coronary disease.
Postconditioning was initially proposed to act by reducing neutrophil-mediated damage in postischemic myocardium,11,20 but this cannot be the sole mechanism of action, because it is protective in isolated perfused hearts21,23 and cell culture systems24 that are neutrophil-free. There may be effects to reduce reactive oxygen species production19,20,25 and calcium overload.24 Moreover, emerging evidence indicates that protection may be dependent on adenosine receptor stimulation,23 opening of mitochondrial KATP channels,22 activation of the prosurvival kinases PI3K-Akt21,23,26 and MEK1/2-Erk1/2,22 and inhibition of mitochondrial permeability transition pore opening,27 key factors implicated in ischemic preconditioning–induced protection. These observations suggest that postconditioning and ischemic preconditioning have common signaling pathways, which may explain why these protective phenomena are equally effective in protecting against IR injury.11,19,21
In humans, preservation of FMD by postconditioning is consistent with increased nitric oxide availability during reperfusion, either due to decreased nitric oxide inactivation11,24 and/or increased nitric oxide synthesis.21 In the present model, IR-induced endothelial injury resolves spontaneously within 60 minutes of reperfusion,16 consistent with "endothelial stunning" rather than necrosis in response to 20 minutes of ischemia. This may explain the complete abrogation of endothelial dysfunction caused by postconditioning that we observed. Whether the postconditioning schedule we used will protect from more substantial endothelial injury (as would occur in the clinical setting of acute myocardial infarction) or be protective in patients with preexisting endothelial dysfunction (as occurs in patients with risk factors for atherosclerosis) remains to be determined.
In summary, our data demonstrate an effect of postconditioning to prevent endothelial IR injury in the human forearm vasculature. The data indicate that much of the endothelial dysfunction caused by sublethal IR injury occurs during reperfusion. Moreover, mechanisms activated in the immediate phase of reperfusion are necessary for endothelial IR injury. These data support the idea that the reperfusion phase of IR injury is a legitimate therapeutic target. Given the increasing role of primary intervention in the treatment of acute coronary syndromes, postconditioning schedules or pharmacological manipulation may modify reperfusion injury and reduce infarct size sufficiently to improve outcomes.28
Acknowledgments
We would like to acknowledge the British Heart Foundation for supporting our research.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Disclosures
None.
References
Jennings RB, Reimer KA. Factors involved in salvaging ischemic myocardium: effect of reperfusion of arterial blood. Circulation. 1983; 68: I-25–I-36.
Zahn R, Schiele R, Schneider S, Gitt AK, Wienbergen H, Seidl K, Voigtlander T, Gottwik M, Berg G, Altmann E, Rosahl W, Senges J. Primary angioplasty versus intravenous thrombolysis in acute myocardial infarction: can we define subgroups of patients benefiting most from primary angioplasty Results from the pooled data of the Maximal Individual Therapy in Acute Myocardial Infarction Registry and the Myocardial Infarction Registry. J Am Coll Cardiol. 2001; 37: 1827–1835.
Braunwald E, Kloner RA. Myocardial reperfusion: a double-edged sword J Clin Invest. 1985; 76: 1713–1719.
Heyndrickx GR, Millard RW, McRitchie RJ, Maroko PR, Vatner SF. Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J Clin Invest. 1975; 56: 978–985.
Bolli R, Marban E. Molecular and cellular mechanisms of myocardial stunning. Physiol Rev. 1999; 79: 609–634.
Matsumura K, Jeremy RW, Schaper J, Becker LC. Progression of myocardial necrosis during reperfusion of ischemic myocardium. Circulation. 1998; 97: 795–804.
Eefting F, Rensing B, Wigman J, Pannekoek WJ, Liu WM, Cramer MJ, Lips DJ, Doevendans PA. Role of apoptosis in reperfusion injury. Cardiovasc Res. 2004; 61: 414–426.
Okamoto F, Allen BS, Buckberg GD, Bugyi H, Leaf J. Reperfusion conditions: importance of ensuring gentle versus sudden reperfusion during relief of coronary occlusion. J Thorac Cardiovasc Surg. 1986; 92: 613–620.
Sato H, Jordan JE, Zhao ZQ, Sarvotham SS, Vinten-Johansen J. Gradual reperfusion reduces infarct size and endothelial injury but augments neutrophil accumulation. Ann Thorac Surg. 1997; 64: 1099–1107.
Vinten-Johansen J, Zhao ZQ, Zatta AJ, Kin H, Halkos ME, Kerendi F. Postconditioning: a new link in nature’s armor against myocardial ischemia-reperfusion injury. Basic Res Cardiol. 2005; 100: 295–310.
Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003; 285: H579–H588.
Tsang A, Hausenloy DJ, Yellon DM. Myocardial postconditioning: reperfusion injury revisited. Am J Physiol Heart Circ Physiol. 2005; 289: H2–H7.
Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L’Huillier I, Aupetit JF, Bonnefoy E, Finet G, Andre-Fouet X, Ovize M. Postconditioning the human heart. Circulation. 2005; 112: 2143–2148.
Loukogeorgakis SP, Panagiotidou AT, Broadhead MW, Donald A, Deanfield JE, MacAllister RJ. Remote ischemic preconditioning provides early and late protection against endothelial ischemia reperfusion injury in humans: role of the autonomic nervous system. J Am Coll Cardiol. 2005; 46: 450–456.
Kharbanda RK, Mortensen UM, White PA, Kristiansen SB, Schmidt MR, Hoschtitzky JA, Vogel M, Sorensen K, Redington AN, MacAllister R. Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation. 2002; 106: 2881–2883.
Kharbanda RK, Peters M, Walton B, Kattenhorn M, Mullen M, Klein N, Vallance P, Deanfield J, MacAllister R. Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans in vivo. Circulation. 2001; 103: 1624–1630.
Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, MacAllister RJ. Heterogenous nature of flow-mediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia. Circ Res. 2001; 88: 145–151.
Sorensen KE, Celermajer DS, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Thomas O, Deanfield JE. Non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility. Br Heart J. 1995; 74: 247–253.
Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS, Sun HY, Guyton RA, Vinten-Johansen J, Zhao ZQ. Myocardial protection with postconditioning is not enhanced by ischemic preconditioning. Ann Thorac Surg. 2004; 78: 961–969.
Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, Kerendi F, Guyton RA, Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res. 2004; 62: 74–85.
Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res. 2004; 95: 230–232.
Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol. 2004; 44: 1103–1110.
Yang XM, Philipp S, Downey JM, Cohen MV. Postconditioning’s protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res Cardiol. 2005; 100: 57–63.
Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA, Vinten-Johansen J, Zhao ZQ. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. Am J Physiol Heart Circ Physiol. 2005; 288: H1900–H1908.
Serviddio G, Di Venosa N, Federici A, D’Agostino D, Rollo T, Prigigallo F, Altomare E, Fiore T, Vendemiale G. Brief hypoxia before normoxic reperfusion (postconditioning) protects the heart against ischemia-reperfusion injury by preventing mitochondria peroxyde production and glutathione depletion. FASEB J. 2005; 19: 354–361.
Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology. 2005; 102: 102–109.
Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M. Postconditioning inhibits mitochondrial permeability transition. Circulation. 2005; 111: 194–197.
Vinten-Johansen J, Yellon DM, Opie LH Postconditioning: a simple, clinically applicable procedure to improve revascularization in acute myocardial infarction. Circulation. 2005; 112: 2085–2088.(Stavros P. Loukogeorgakis, BSc, PhD; Ann)
Hatter Cardiovascular Institute and Centre for Cardiology (D.M.Y.)
Centre for Clinical Pharmacology and Therapeutics (S.P.L., A.T.P., R.J.M.), University College London, London, United Kingdom.
Abstract
Background— Hypoxic cell death follows interruption of blood supply to tissues. Although successful restoration of blood flow is mandatory for salvage of ischemic tissues, reperfusion can paradoxically place tissues at risk of further injury. Brief periods of ischemia applied at the onset of reperfusion have been shown to reduce ischemia-reperfusion (IR) injury, a phenomenon called postconditioning. The aim of this study was to determine whether postconditioning protects against endothelial IR injury in humans, in vivo.
Methods and Results— Brachial artery endothelial function was assessed by vascular ultrasound to measure flow-mediated dilation (FMD) in response to forearm reactive hyperemia. FMD was measured before and after IR (20 minutes of arm ischemia followed by 20 minutes of reperfusion) in healthy volunteers. To test the protective effects of postconditioning, 3 cycles of reperfusion followed by ischemia (each lasting 10 or 30 seconds) were applied immediately after 20 minutes of arm ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before the application of the 10-second postconditioning stimulus. IR caused endothelial dysfunction (FMD 9.1±1.2% pre-IR, 3.6±0.7% post-IR, P<0.001; n=11), which was prevented by postconditioning applied as 10-second cycles of reperfusion/ischemia (FMD 9.9±1.7% pre-IR, 8.3±1.4% post-IR, P=NS; n=11) and 30-second cycles of reperfusion/ischemia (FMD 10.8±1.7% pre-IR, 9.5±1.5% post-IR, P=NS; n=10) immediately at the onset of reperfusion. No protection was observed when the application of the 10-second postconditioning stimulus was delayed for 1 minute after the onset of reperfusion (FMD 9.8±1.2% pre-IR, 4.0±0.9% post-IR, P<0.001; n=8).
Conclusions— This study demonstrates for the first time that postconditioning can protect against endothelial IR injury in humans. Postconditioning might reduce tissue injury when applied at the onset of reperfusion by modifying the reperfusion phase of IR.
Key Words: endothelium ischemia reperfusion reperfusion injury postconditioning
Introduction
The prerequisite for salvage of viable myocardium and limitation of infarct size after an acute ischemic event is timely reperfusion.1,2 However, restoration of coronary blood flow carries with it the risk of further myocardial injury (reperfusion injury).3 Events occurring during reperfusion have been shown to be responsible in part for reversible (stunning) and, more important, lethal (necrosis/apoptosis) injury in animal models.4–7 Although the contribution of reperfusion injury to infarct size in humans is unknown, it is possible that minimizing reperfusion injury might reduce infarct size in humans.
Clinical Perspective p 1019
Modulating blood flow on reperfusion (through gradual or intermittent reperfusion) has been shown to reduce experimental infarct size.8,9 In particular, Vinten-Johansen et al10 have shown that a schedule of intermittent reperfusion (termed "postconditioning") applied at the onset of reperfusion reduces infarct size in animal models of myocardial ischemia-reperfusion (IR) injury. The protective effect of postconditioning is of a similar magnitude to that seen with ischemic preconditioning.11 However, unlike preconditioning, the influence of postconditioning is restricted to the reperfusion phase of IR injury, and this has renewed interest in the reperfusion phase as a target for cardioprotection.12
Staat et al13 have recently shown that a postconditioning protocol of intermittent reperfusion reduces myocardial injury during primary angioplasty in patients with acute myocardial infarction. We have developed a model of IR injury that results in endothelial dysfunction in conduit14 and resistance vessels.15 In the present study, we used this model to determine the potential protective effects of different schedules of ischemic postconditioning against endothelial IR injury in humans, in vivo.
Methods
Subjects
Forty studies were performed on 11 healthy volunteers (6 men, 5 women; mean age±SD 23.6±4.2 years; range 18 to 33 years). All volunteers gave informed consent. Studies were approved by the local research ethics committee and performed in a temperature-controlled laboratory (24°C to 26°C). All studies repeated in the same volunteers were at least 7 days apart.
Induction of IR
The nondominant arm was made ischemic by the inflation of a 9-cm-wide blood pressure cuff placed around the upper part of the arm to a pressure of 200 mm Hg for 20 minutes, as described previously.14,16
Induction of Postconditioning
Postconditioning was induced by the application of intermittent short periods of ischemia and reperfusion on the nondominant arm early during reperfusion. After index ischemia, the arm was allowed to reperfuse for 10 seconds, after which the blood pressure cuff was inflated again to 200 mm Hg, which made the arm ischemic for 10 seconds. This deflation/inflation cycle was repeated a total of 3 times (1 minute total duration; PostC10). The effect of longer cycles of postconditioning (3 cycles of 30 seconds of reperfusion alternating with 30 seconds of ischemia) was also determined (PostC30).
Assessment of Conduit Vessel Endothelial Function
Endothelial function of the brachial artery was assessed by flow-mediated dilation (FMD) of the brachial artery in the nondominant arm, as described previously.17 In this technique, reactive hyperemia of the forearm is used to increase blood flow in the brachial artery, resulting in brachial artery dilatation. A B-mode scan of the brachial artery was obtained in longitudinal section between 5 and 10 cm above the antecubital fossa with a 7.0-MHz linear-array transducer (spatial resolution of 0.1 mm18) and a standard Acuson XP10 system. Longitudinal, ECG-gated, end-diastolic images were acquired every 3 seconds for offline analysis. Arterial diameter over a 1- to 2-cm segment was determined for each image with the use of automatic edge-detection software (Brachial Tools, Medical Imaging Applications). Blood flow was manipulated in the brachial artery by a 7-cm-wide blood pressure cuff placed around the forearm immediately below the antecubital fossa. After 1 minute of baseline flow, the cuff was inflated to 300 mm Hg for 5 minutes and released, which resulted in a brief episode of reactive hyperemia. Brachial artery diameter changes in response to blood flow were assessed for an additional 5 minutes. Blood flow velocity was monitored continuously by pulsed-wave Doppler.
Experimental Protocols
Effect of IR on Vascular Dilator Function
To determine the effect of IR on endothelial function, FMD was assessed before ischemia (baseline) and at 20 minutes after reperfusion (n=11; Figure 1, protocol a). We have previously demonstrated that this protocol results in brachial artery endothelial dysfunction but does not have an effect on vascular smooth muscle function, because it did not alter the dilator response to nitroglycerin (NTG).14
Effect of Postconditioning on Endothelial IR Injury
To establish that protection against endothelial IR can be achieved by modifying reperfusion, postconditioning was induced in the same group of volunteers. Three cycles of 10 seconds of reperfusion and 10 seconds of ischemia (PostC10; n=11; Figure 1, protocol b) or 3 cycles of 30 seconds of reperfusion and 30 seconds of ischemia (PostC30; n=10; Figure 1 protocol c) were applied immediately after index ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before application of the 10-second-cycle postconditioning stimulus (DelayedPostC10; n=8; Figure 1, protocol d).
Calculations and Statistics
All data are expressed as mean±SE unless otherwise stated. Brachial artery diameter was measured in millimeters and dilation expressed as percentage increase from baseline diameter. The FMD flow stimulus during reactive hyperemia was expressed as the ratio of peak to baseline volume flow per minute. Data were compared by paired Student t test or repeated-measures ANOVA as appropriate. For multiple comparisons (8 groups), probability values by ANOVA were Bonferroni adjusted. In all cases, P<0.05 was considered statistically significant.
Results
All subjects tolerated the procedures without any complications. There were no differences in the responses between men and women.
Effect of IR on Vascular Dilator Function
The IR protocol had no effect on blood pressure and heart rate (Table). IR reduced brachial artery FMD (9.1±1.2% pre-IR versus 3.6±0.7% post-IR, P<0.001, ANOVA; n=11; Figure 2a), which is consistent with endothelial dysfunction. These results could not be explained by differences in brachial artery diameter (3.4±0.2 mm pre-IR versus 3.4±0.2 mm post-IR, P=NS, t test) and FMD flow stimulus (peak to baseline volume flow ratio 11.3±1.5 pre-IR versus 10.9±1.0 post-IR, P=NS, t test; Table).
Summary of Pre- and Post-IR Data From Sequential Studies in Healthy Volunteers
Effect of Postconditioning on Endothelial IR Injury
Postconditioning had no effect on blood pressure, heart rate, arterial diameter, or flow stimulus during reactive hyperemia (Table). Ten-second-cycle postconditioning (PostC10), applied immediately at the onset of reperfusion, prevented IR-induced endothelial dysfunction (FMD 9.9±1.7% pre- versus 8.3±1.4% post-IR+PostC10, P=NS, ANOVA; n=11; Figure 2b). Similarly, 30-second-cycle postconditioning (PostC30) was also protective (FMD 10.8±1.7% pre- versus 9.5±1.5% post-IR+PostC30, P=NS, ANOVA; n=10; Figure 2c). However, no protection was observed when the application of the PostC10 stimulus was delayed for 1 minute after blood flow restoration to the arm (FMD 9.8±1.2% pre- versus 4.0±0.9% post-IR+DelayedPostC10, P<0.001, ANOVA; n=8; Figure 2d). Mean post-IR FMD in PostC10 and PostC30 studies was significantly higher than the respective values obtained in control (IR alone; P<0.001, ANOVA) and DelayedPostC10 experiments (P<0.001, ANOVA; Table).
Discussion
This study demonstrates for the first time in humans in vivo that ischemic postconditioning prevents endothelial IR injury in conduit vessels. The first moments of reperfusion are critical for endothelial protection by postconditioning, because the protective effect disappeared when the stimulus was applied 1 minute after the onset of reperfusion.
Ischemic postconditioning describes protection against IR injury by a series of brief interruptions of blood flow applied at the onset of reperfusion. Studies by Zhao et al11 and Halkos et al19 demonstrated in a canine model of myocardial infarction that the application of a postconditioning protocol immediately after coronary artery occlusion reduced myocardial infarct size, an effect comparable to that of ischemic preconditioning. Since then, the cardioprotective effects of postconditioning have been confirmed in the in vivo and ex vivo heart in rats20,21 and rabbits,22,23 as well as in isolated rat cardiomyocytes.24 More recently, Staat et al13 have demonstrated that postconditioning reduces infarct size in patients undergoing primary angioplasty for acute myocardial infarction.
In the present study, we used arm ischemia to model IR injury in humans. Previously, we have shown that ischemia causes transient endothelial dysfunction of the brachial artery, with preservation of smooth muscle function.14 This model is a convenient way to compare different schedules of postconditioning stimuli. In our IR model, the degree of protection by postconditioning is similar to that achieved by ischemic preconditioning and remote ischemic preconditioning in previous studies in healthy volunteers.14,16 This is in agreement with animal data19,21 and suggests that injury suffered during reperfusion is largely responsible for the IR-induced endothelial dysfunction we observed. Moreover, our present findings demonstrate that postconditioning induces protection against endothelial IR injury by modifying events that occur during the early stages of reperfusion20; a delay in the application of the postconditioning stimulus for as little as 1 minute resulted in loss of protection. Cycle length (10 versus 30 seconds) mattered less than the timing of the initiation of the postconditioning schedule. In this regard, it is worth noting that the recent study of primary angioplasty in patients used a postconditioning protocol of four 60-second cycles of ischemia/reperfusion, also initiated within the first minute of reperfusion.13 It remains to be determined to what extent these results in the brachial vasculature can be extrapolated to the coronary vasculature. Nonetheless, the present study provides corroborative evidence that postconditioning occurs in humans and has similar characteristics to those described in animal models and patients with acute coronary disease.
Postconditioning was initially proposed to act by reducing neutrophil-mediated damage in postischemic myocardium,11,20 but this cannot be the sole mechanism of action, because it is protective in isolated perfused hearts21,23 and cell culture systems24 that are neutrophil-free. There may be effects to reduce reactive oxygen species production19,20,25 and calcium overload.24 Moreover, emerging evidence indicates that protection may be dependent on adenosine receptor stimulation,23 opening of mitochondrial KATP channels,22 activation of the prosurvival kinases PI3K-Akt21,23,26 and MEK1/2-Erk1/2,22 and inhibition of mitochondrial permeability transition pore opening,27 key factors implicated in ischemic preconditioning–induced protection. These observations suggest that postconditioning and ischemic preconditioning have common signaling pathways, which may explain why these protective phenomena are equally effective in protecting against IR injury.11,19,21
In humans, preservation of FMD by postconditioning is consistent with increased nitric oxide availability during reperfusion, either due to decreased nitric oxide inactivation11,24 and/or increased nitric oxide synthesis.21 In the present model, IR-induced endothelial injury resolves spontaneously within 60 minutes of reperfusion,16 consistent with "endothelial stunning" rather than necrosis in response to 20 minutes of ischemia. This may explain the complete abrogation of endothelial dysfunction caused by postconditioning that we observed. Whether the postconditioning schedule we used will protect from more substantial endothelial injury (as would occur in the clinical setting of acute myocardial infarction) or be protective in patients with preexisting endothelial dysfunction (as occurs in patients with risk factors for atherosclerosis) remains to be determined.
In summary, our data demonstrate an effect of postconditioning to prevent endothelial IR injury in the human forearm vasculature. The data indicate that much of the endothelial dysfunction caused by sublethal IR injury occurs during reperfusion. Moreover, mechanisms activated in the immediate phase of reperfusion are necessary for endothelial IR injury. These data support the idea that the reperfusion phase of IR injury is a legitimate therapeutic target. Given the increasing role of primary intervention in the treatment of acute coronary syndromes, postconditioning schedules or pharmacological manipulation may modify reperfusion injury and reduce infarct size sufficiently to improve outcomes.28
Acknowledgments
We would like to acknowledge the British Heart Foundation for supporting our research.
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Disclosures
None.
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