Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion: Role of Hydrogen Peroxide

Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion Role of Hydrogen Peroxide Naris Thengchaisri, Robert Shipley, Yi Ren, Janet Parker, Lih Kuo Objective—Exercise training has been shown to restore vasodilation to nitric oxide synthase (NOS) activation in arterioles distal to coronary artery occlusion. Because reactive oxygen species are generated during NOS uncoupling and the production of vasodilator H2O2 is increased during exercise in patients with coronary disease, we proposed that H2O2 may contribute to the restoration of vasodilation in porcine coronary occlusion model. Methods and Results—Left circumflex (LCX) coronary artery of miniature swine was progressively occluded for 8 weeks followed by exercise training (EX; 5 days/wk treadmill) or sedentary (SED) protocols for 12 weeks. Arterioles were isolated from distal LCX and nonoccluded left anterior descending (LAD) artery for in vitro study. Vasodilation to NOS activators adenosine and ionomycin was impaired in SED LCX, but not LAD, arterioles. This impairment was restored by L-arginine. NO production induced by adenosine was also reduced in SED LCX arterioles. EX had no effect on LAD arterioles but improved NO production and restored dilation of LCX arterioles. NOS blockade (L-NAME) inhibited vasodilation to NOS activators in LAD (SED & EX) arterioles but was ineffective in SED LCX arterioles. In EX LCX arterioles, vasodilation to NOS activators was slightly inhibited by L-NAME but abolished by catalase. H2O2 production was markedly increased by adenosine in EX LCX arterioles. Conclusions—This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H2O2, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion. (Arterioscler Thromb Vasc Biol. 2007;27:791-798.) Key Words: nitric oxide 䡲 vasodilation 䡲 collateral circulation 䡲 hydrogen peroxide 䡲 L-arginine

E

ndothelial dysfunction has been documented in the collateral-dependent coronary microcirculation by impaired vasorelaxation to endothelium-dependent agonists such as adenosine dinucleotide phosphate (ADP)1 and bradykinin.1,2 On the other hand, our laboratory recently demonstrated that exercise training enhances vasodilation of collateral-dependent coronary arterioles to bradykinin2 and vascular endothelial growth factor (VEGF).3 Traininginduced vascular effects appear due, at least in part, to increases in endothelial nitric oxide synthase (eNOS) mRNA levels2 and nitric oxide (NO) production.2,3 Although recent studies suggest that endothelium-derived hyperpolarizing factor(s) (EDHF) may play a compensatory role in NO-mediated vasodilatory function in the presence of coronary vascular diseases,4 the potential involvement of EDHF in effects of exercise training on collateral-dependent vascular function has not been clarified. Hydrogen peroxide (H2O2), has been proposed to be EDHF in human mesenteric

arteries5 and porcine coronary microvessels6 and can be involved in vasorelaxation during uncoupling of NO synthesis.7,8 Recent studies in atrial coronary arterioles isolated from heart disease patients indicate a release of H2O2 in response to increased flow4; furthermore, plasma H2O2 levels are increased during bouts of exercise in patients with coronary heart disease.9 Because H2O2 is diffusible and stable, and its production and removal are highly regulated, this molecule has been implicated as a second messenger in a variety of intracellular responses10,11 and may play an important role in vasoregulation under pathophysiological conditions.4 Herein, we hypothesized that H2O2 may contribute to the exercise training-induced restoration of vasodilation to NOS activation in the collateral-dependent microcirculation. To test this hypothesis, receptor-dependent (adenosine) and receptorindependent (ionomycin), NO-mediated vasodilations were evaluated in sedentary and exercise-trained animals to determine the potential involvement of H2O2 in vasodilation to

Original received October 17, 2006; final version accepted January 8, 2007. From the Department of Systems Biology and Translational Medicine, Cardiovascular Research Institute, College of Medicine, Texas A&M Health Science Center, Temple, Tex. Current address for N.T.: Faculty of Veterinary Medicine, Kasetsart University, 50 Paholyothin Road, Bangkhen, Bangkok 10900, Thailand. Correspondence to Lih Kuo, PhD, Department of Systems Biology and Translational Medicine, Cardiovascular Research Institute, College of Medicine, Texas A&M Health Science Center, 702 Southwest H.K. Dodgen Loop, Temple, TX 76504. E-mail [email protected] © 2007 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

DOI: 10.1161/01.ATV.0000258416.47953.9a

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NOS activation in collateral-dependent coronary arterioles distal to coronary artery occlusion.

Methods Porcine Model of Chronic Coronary Occlusion and Collateral Development Ameroid-induced progressive coronary occlusion was performed as described previously.2,12–15 Briefly, adult female Yucatan miniature swine (25 to 45 kg; Sinclair Research Center, Columbia, Mo) were sedated with ketamine (20 mg/kg; i.m.), midazolam (0.5 mg/kg; i.m.), and glycopyrrolate (0.004 mg/kg; i.m.) and then intubated and maintained with 3% isoflurane and 97% O2 during the sterile surgical procedure. An ameroid constrictor was placed around the proximal left circumflex (LCX) coronary artery. Ameroid occlusion is successful in more than 95% of animals; however, because of coronary collateral development, this procedure results in minimal myocardial infarction.1,12,15 Protocols were approved by Texas A&M Institutional Animal Care and Use Committee.

Exercise-Training Program Coronary-occluded animals were allowed 2 months for recuperation and collateral development before random division into equal groups of either sedentary (SED; n⫽16) or exercise-trained (EX; n⫽18) groups. EX animals entered a progressive treadmill running program for 12 weeks, and SED animals remained inactive by pen confinement during an equivalent period. The training protocol has been used extensively by this laboratory2,3,16 and others.17,18 In brief, during the first week of training, animals ran at 4 to 5 mph, 0% grade for 15 minutes (speed run) and 3 mph, 0% grade for 20 to 30 minutes (endurance). Speed and duration were progressively increased depending on the ability of the animal. By week 12 of training, animals ran 85 minutes/d, 5 days/week. This consisted of a 5-minute warm-up (2.5 mph), a 15-minute (6 mph), a 60-minute run 4.5 to 5 mph, and a 5-minute cool-down (2.5 mph).

Isolation and Preparation of Coronary Arterioles After completion of EX or SED protocols, pigs were sedated with ketamine (25 mg/kg; i.m.) and xylazine (2.25 mg/kg; i.m.) and anesthetized with thiopental sodium (10 mg/kg; i.v.). After removal of the heart, size-matched arterioles (⬇100 ␮m in internal diameter in situ) were isolated from the collateral-dependent myocardium (distal to LCX coronary occlusion) and the myocardium perfused by the nonoccluded left anterior descending (LAD) coronary artery for in vitro study as previously described.19 –21 After isolation, vessels were cannulated with glass micropipettes and pressurized to 60 cm H2O intraluminal pressure. The cannulated vessel was bathed in physiological salt solution (PSS) containing bovine serum albumin (BSA; 1%, USB) at 37°C.19 –21 Internal diameters of the vessel were measured throughout the experiment using video microscopic techniques incorporated into the MacLab (ADInstruments Inc) data acquisition system.19 Arterioles were allowed 1 hour of stabilization for the development of stable basal tone before analysis of vasomotor function.

Experimental Protocols The NO-mediated vasodilations in response to receptor-dependent and -independent agonists, adenosine (0.1 nmol/L to 10 ␮mol/L)20 and ionomycin (0.1 nmol/L to 1 ␮mol/L),21 respectively, were examined. The vasodilations in response to the direct activation of smooth muscle ATP-sensitive potassium (KATP) channel and guanylyl cyclase by pinacidil (0.3 to 3 ␮mol/L) and sodium nitroprusside (1 nmol/L to 10 ␮mol/L), respectively, were also evaluated. Selected experiments were performed after treating the vessels with NG-nitroL-arginine methyl esterase (L-NAME; 10 ␮mol/L), catalase (1,000 U/mL), or L-arginine (3 mmol/L) for 30 minutes. All drugs were applied abluminally. Measurements were made and recorded 2 to 3 minutes after drug administration, when the response was stabilized. Order of drug administration was random. Vessels were washed with PSS and allowed to equilibrate in PSS for 20 to 30 minutes between

interventions. At the end of each experiment, vessels were relaxed with sodium nitroprusside (100 ␮mol/L) to obtain maximal diameter21 at 60 cm H2O intraluminal pressure.

NO Assay NO production from coronary arterioles (5 to 7 vessels/sample, ⬇100 ␮m inner diameter, 1 to 2 mm in length) was evaluated by measuring nitrite using a chemiluminescence NO analyzer (Siever Instruments) as described previously.20 Vessels were incubated (30 minutes) in a microcentrifuge tube (100 ␮L PSS, 37°C) without or with 1 ␮mol/L adenosine for basal and stimulated NO production, respectively. Background nitrite in the PSS was subtracted from the sampled solution to obtain NO production. Protein levels in each tube were quantified by bicinchoninic acid protein assay (Pierce) and used as a basis to normalize the NO production.

H2O2 Detection Coronary arterioles were subjected to H2O2 measurement based on the fluorescence detection of dichlorofluorescein (DCF), which is formed by H2O2 oxidation of the nonfluorescent precursor 2⬘,7⬘-dichlorodihydrofluorescein diacetate (DCFH).4 Isolated coronary arterioles were incubated with PSS (control) or adenosine (1 ␮mol/L), in the absence or presence of catalase (1000 U/mL), at 37°C for 30 minutes. Vessels were then loaded with DCFH (5 ␮mol/L) for 10 minutes. After washing, vessels were embedded in OCT compound and subjected to cryosection (10 ␮m thickness). Fluorescence images were acquired at 525 nm by a fluorescence microscope (Nikon Diaphot 300). Control and experimental tissues were placed on the same slide and processed under the same settings for image acquisition.

Chemicals Drugs were obtained from Sigma, except as otherwise stated. Adenosine, catalase, L-arginine, L-NAME, and sodium nitroprusside were dissolved in PSS. Pinacidil was dissolved in ethanol, and ionomycin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (10 mmol/L). Subsequent concentrations of pinacidil and ionomycin were diluted in PSS. Final concentrations of ethanol and DMSO in the vessel bath were less than 0.1 and 0.01%, respectively. Vehicle control studies indicated that the final concentration of ethanol and DMSO had no effect on arteriolar function.

Data Analysis Diameter changes in response to agonists were normalized to the vasodilation induced by 100 ␮mol/L sodium nitroprusside and expressed as a percentage of maximal dilation.21 All data are presented as means⫾SEM. Statistical comparisons of vasomotor responses under different treatments were performed with two-way ANOVA and tested with Fisher’s protected least significant difference multiple-range test. Differences in resting diameter before and after pharmacological interventions were compared by paired Student t test. Probability values ⬍0.05 were considered to be significant.

Results Resting Tone and Arteriolar Dilation In the present study, the average maximal internal diameters for arterioles from nonoccluded LAD and occluded LCX regions of SED pigs were 93⫾9 ␮m and 111⫾9 ␮m, respectively. In EX pigs, the average maximal diameters for LAD and LCX arterioles were 115⫾7 ␮m and 112⫾6 ␮m, respectively. The average maximum diameters of coronary arterioles were comparable between SED and EX pigs. The resting tone (60% to 65% of maximal diameter) was comparable in each vessel group. Vasodilation of SED LCX arterioles to adenosine (Figure 1A) and ionomycin (Figure 1B) was significantly reduced compared with SED LAD

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Figure 1. Coronary arteriolar dilation to adenosine and ionomycin. Dilation of coronary arterioles to adenosine (A) and ionomycin (B) from collateraldependent (LCX) regions of sedentary (SED) animals was significantly attenuated in comparison to that from nonoccluded (LAD) regions. Exercise training (EX) restored vasodilation to adenosine (C) and ionomycin (D). Number of vessel studied is indicated in parenthesis. *P⬍0.05 between groups.

arterioles. Although exercise training had no effect on the dilation of LAD arterioles to adenosine (Figure 1C) and ionomycin (Figure 1D), the vasodilations to these agonists were improved in LCX arterioles and the extent of these dilations was identical to that in SED (Figure 1A and 1B) or EX (Figure 1C and 1D) LAD arterioles. Impaired vasodilation to adenosine and ionomycin in SED LCX arterioles appears to involve altered endothelial function since vasomotor responses of these vessels to the activation of smooth muscle KATP channel and guanylyl cyclase by pinacidil (supplemental Figure IA, available online at http://atvb. ahajournals.org) and sodium nitroprusside (supplemental Figure IB), respectively, were similar between SED LCX and SED LAD arterioles. Furthermore, exercise training had no effect on the dilation of these vessels to sodium nitroprusside (supplemental Figure IB).

Role of NO-Mediated Vasodilation L-NAME significantly inhibited dilations of SED LAD and EX LAD arterioles to adenosine (Figure 2A) and ionomycin (Figure 2B) in an identical manner. In contrast, L-NAME failed to inhibit dilations to adenosine and ionomycin in SED LCX arterioles (Figure 2C and 2D), suggesting loss of NO-mediated relaxation in collateral-dependent SED LCX arterioles. Furthermore, L-NAME only produced a slight inhibitory effect on adenosine- and ionomycin-induced dilation of EX LCX arterioles (Figure 2C and 2D) compared with that observed in nonoccluded LAD arterioles (Figure 2A and 2B).

NO Production To further support the functional data, NO production from LAD and LCX arterioles from SED and EX groups was

measured. Basal NO production was not significantly different in arterioles from SED (LAD: 100⫾38 nmol/g protein, LCX: 110⫾35 nmol/g protein) and EX (LAD: 122⫾32 nmol/g protein, LCX: 142⫾28 nmol/g protein) animals (n⫽6). Adenosine (1 ␮mol/L) significantly increased NO production in SED (173⫾32% above basal level) and EX (182⫾38% above basal level) LAD arterioles (n⫽6, P⬍0.05). However, adenosine-stimulated NO production was reduced in SED LCX arterioles (38⫾20% above basal level). In EX LCX arterioles, NO production in response to adenosine stimulation was increased (92⫾21% above basal level), but the extent was significantly lower than SED or EX LAD groups (n⫽6, P⬍0.05). Interestingly, there was no difference in vasodilatory response to adenosine between arterioles from EX LAD and EX LCX regions (Figure 1C and 1D). These data may suggest that other mediator(s) in addition to NO may be responsible for restored vasodilator function of EX LCX arterioles after coronary occlusion.

Role of H2O2 in Vasodilation Catalase did not alter vasodilation of LAD (SED or EX) arterioles (Figure 3A and 3B) or SED LCX arterioles (Figure 3C and 3D) in response to adenosine and ionomycin. However, catalase significantly attenuated vasodilation of EX LCX arterioles to adenosine (Figure 3C) and ionomycin (Figure 3D), indicating involvement of H2O2 in these responses. In the presence of L-arginine (3 mmol/L), the inhibitory effect of catalase was not observed (Figure 3C and 3D). The LAD and LCX arterioles isolated from EX animals also dilated to exogenous H2O2 in a dose-dependent manner and there was no difference in these dilations (supplemental

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Figure 2. Effect of NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation to adenosine and ionomycin. L-NAME significantly inhibited dilation of LAD arterioles to adenosine (A) and ionomycin (B) in both sedentary (SED) and exercise-trained (EX) pigs. L-NAME significantly attenuated EX LCX, but had no effect on SED LCX dilation in response to adenosine (C) and ionomycin (D). Number of vessel studied is indicated in parenthesis. *P⬍0.05 between groups.

Figure II). H2O2-induced dilations were inhibited by catalase (1,000 U/mL; supplemental Figure II). Catalase did not change basal vascular tone or vasorelaxation to sodium nitroprusside (data not shown).

Effects of Exogenous L-Arginine L-Arginine (3 mmol/L) did not alter vascular tone or vasodilations to adenosine (Figure 4A) and ionomycin (Figure 4B) in coronary arterioles isolated from LAD region. How-

Figure 3. Effect of catalase on concentrationdependent responses to adenosine and ionomycin. Catalase did not alter vasodilation of LAD arterioles from both sedentary (SED) and exercisetrained (EX) pigs in response to adenosine (A) and ionomycin (B). However, catalase significantly inhibited the vasodilation of EX LCX arterioles in response to adenosine (C) and ionomycin (D). These inhibitory effects were reversed by treating the vessels with L-arginine (L-Arg). Number of vessel studied is indicated in parenthesis. *P⬍0.05 between groups.

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Figure 4. Effect of L-arginine on concentrationdependent dilations in response to adenosine and ionomycin. In nonoccluded (LAD) coronary arterioles isolated from sedentary (SED) animals, L-arginine did not alter vasodilation of to adenosine (A) and ionomycin (B). However, L-arginine significantly enhanced the vasodilation of collateral-dependent (LCX) arterioles in response to adenosine (C) and ionomycin (D). These enhancements were abolished by treating the vessels with L-NAME. Number of vessel studied is indicated in parenthesis. *P⬍0.05 vs SED LCX

ever, L-arginine significantly enhanced vasodilation of LCX arterioles in response to adenosine (Figure 4C, n⫽5) and ionomycin (Figure 4D, n⫽5). These enhancements were reversed by L-NAME (Figure 4C and 4D, n⫽3) but not by catalase (n⫽2, data not shown). In addition, the NO production in response to adenosine stimulation was significantly increased in the presence of L-arginine (control: 28⫾18% above basal level versus L-arginine: 130⫾35% above basal level; n⫽4, P⬍0.05), but the extent was slightly lower than that produced by SED or EX LAD arterioles (ie, about 180% above basal level). Although vasodilation of SED LCX arterioles to adenosine and ionomycin was enhanced by L-arginine, dilation remained slightly less than arterioles from SED LAD region (Figure 4; EC50 for adenosine: 1.8⫾0.1⫻10⫺7 mol/L versus 7.4⫾1.4⫻10⫺8 mol/L, P⬍0.05; EC50 for ionomycin: 6.5⫾1.6⫻10⫺7 mol/L versus 3.2⫾0.7⫻10⫺7 mol/L, P⬍0.05). L-Arginine did not affect vasodilation in response to sodium nitroprusside (data not shown, n⫽3).

Production of H2O2

Adenosine (1 ␮mol/L) significantly increased DCF fluorescence signals in EX LCX arterioles and this increase was inhibited by catalase (Figure 5A). In contrast, adenosine did not increase DCF fluorescence signals in EX LAD arterioles (Figure 5B).

Discussion Present studies document that NO-mediated vasodilations in response to adenosine and ionomycin are impaired in collateral-dependent arterioles distal to chronic coronary occlusion; chronic exercise training restores impaired vasodilation responses to these agents. Importantly, we report the

novel finding that the release of H2O2 contributes to the training-induced restoration of endothelium-dependent vasodilation of collateral-dependent microvasculature. In addition, supplementation with L-arginine improves endotheliumdependent NO-mediated dilations of arterioles from the collateral dependent region but not from the nonoccluded region of SED animals. These findings provide new insight into potential mechanisms underlying improved blood flow of collateralized myocardium after exercise training in experimental models15,22 and in human patients with coronary artery disease,23–25 as well as the well-documented beneficial effects of L-arginine in various cardiovascular diseases in human26,27 and animal models.28 –30 Impaired endothelium-dependent vasorelaxation1,13,16,31 and enhanced vasoconstrictor13 responses of coronary vasculature from collateral-dependent myocardial regions have been previously reported. Exercise training appears to improve relaxation of conduit-sized collateral-dependent epicardial arteries in response to bradykinin, ADP, and adenosine.16,31 In the present study, we extend previous findings and demonstrate, for the first time, that chronic coronary artery occlusion impairs NO-mediated vasodilation induced by adenosine and ionomycin in coronary microvasculature. Furthermore, exercise training restores microvascular dilation in response to these agents. Importantly, neither chronic coronary occlusion nor exercise training affected endothelium-independent relaxation to the NO donor sodium nitroprusside, indicating that NO-stimulated downstream cGMP/ PKG cascade in arteriolar smooth muscle was not affected by either intervention. These data are consistent with that reported in larger epicardial coronary arteries using similar animal model.16,31

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Figure 5. Fluorescence images of DCF for H2O2 detection. A, Adenosine (1 ␮mol/L) induced a marked increase in fluorescence signal in arterioles from collateral-dependent (LCX) regions of exercise-trained (EX) animals. In the presence of catalase (1000 U/mL), the increased fluorescence signal was abolished. B, DCF fluorescence signals were not significantly changed by adenosine in arterioles from nonoccluded (LAD) regions of EX animals. Scale bar indicates 50 ␮m. Data shown are representative of 3 separate experiments.

In our previous studies, we have demonstrated that the dilation of porcine coronary arterioles to adenosine is partly mediated by the release of NO from the endothelium via opening of endothelial KATP channels through pertussis toxinsensitive G protein (PTX-G) activation.20 Adenosine, especially at higher concentrations (eg, ␮mol/L range), also activates smooth muscle PTX-G–independent KATP channels for vasodilation through membrane hyperpolarization.20 In the present study, adenosine-induced dilation of collateraldependent coronary arterioles was restored by chronic exercise training; however, the vasodilatory response of arterioles from nonoccluded LAD regions was not enhanced. These results are in agreement with previous reports that exercise training does not enhance adenosine-induced dilation in normal pigs.17 Thus, effects of exercise training on adenosine-induced relaxation responses appear selective for collateral-dependent arterioles. In contrast to the present study, Fogarty et al3 did not detect effects of exercise on vasodilation to adenosine in collateral-dependent arterioles preconstricted with endothelin. This inconsistent result might be related to the use of endothelin in the previous study for maintaining vascular tone.3 Because endothelin has been shown to block vascular KATP channel32,33 possibly through PTX-G inhibition,34 it is likely that endothelin had selectively compromised endothelial pathways necessary for adenosine to exert NOS activation. This view is supported by the observation that NOS inhibitor (L-NMMA) exhibited no effect on adenosine-induced dilation in the previous study.3 It is speculated that the increased release of local endothelin, as commonly observed in myocardial ischemia,35–38 may contribute to the selective endothelial dysfunction related to NO deficiency. In the present study, we found that the dilation of collateral-dependent arterioles to smooth muscle KATP channel activator pinacidil was not altered (supplemental Figure IA), suggesting the lack of effect of coronary occlusion on endothelium-independent dilation of coronary arterioles to adenosine.

Vascular adaptation to chronic exercise training has been demonstrated in various laboratories.2,3,16,17,31,39 However, the mechanism(s) by which exercise training alters endothelial function has not been fully elucidated. Because exercise training did not enhance vascular smooth muscle function in the present study, the machinery responsible for the restoration of endothelium-dependent vasodilation must reside in the endothelium per se. Some studies have implicated an increase in biosynthesis of NO in exercise-induced enhancement of vasorelaxation of coronary arteries16,39 and arterioles2,3,17; these effects have been associated with increased eNOS mRNA levels.2 This contention is partially supported by our observation in the coronary occlusion model because adenosine-stimulated NO production in EX versus SED LCX arterioles was slightly increased and L-NAME moderately attenuated the dilation of EX LCX arterioles in response to NOS activation (Figure 2C and 2D). On the other hand, our data are the first to demonstrate that H2O2 is importantly involved in the mechanisms of enhanced vascular relaxation and that differential roles may be played by NO and H2O2 in the vascular adaptations to chronic coronary occlusion and exercise training. We hypothesize that alteration of complex functions of eNOS plays a pivotal role in this altered signaling mechanism. Stores et al40 recently reported that O2⫺ (precursor of H2O2) can be generated from heme-containing oxygenase domain of eNOS. Although L-NAME has been shown to abolish superoxide formation from neuronal41 and inducible NOS,42 Stores et al40 indicated that O2⫺ production by eNOS remains unaffected by L-NAME despite a complete inhibition of NO production. In the present study, dilation of the EX LCX arterioles to adenosine and ionomycin was less sensitive to L-NAME but highly sensitive to catalase (Figure 3C and 3D). These results indicate that eNOS remains capable of redox activity even in the presence of L-NAME,40 and that increased levels of H2O2 may compensate for decreased NO production during NOS activation. Interest-

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Thengchaisri et al ingly, the inhibitory effect of catalase on EX LCX arteriolar dilations was reversed by L-arginine supplementation (Figure 3C and 3D), suggesting the novel concept of rapid switching between H2O2 and NO production. It is worth noting that Cu/Zn-superoxide dismutase can play an important role in the generation of endothelial H2O243 and improve dilations to the activation of uncoupled eNOS.8 Moreover, exercise training in the pigs has been recently shown to increase Cu/Znsuperoxide dismutase expression in the coronary arterioles.44 It is likely that upregulation of this enzyme by exercise training has contributed to the production of H2O2 for vasodilation in our experimental settings. Indeed, both LAD and LCX arterioles from EX animals were capable of dilating to H2O2 and their responsiveness was rather comparable (supplemental Figure II). However, the elevation of H2O2 by exercise was only observed in the LCX arterioles (Figure 5), suggesting the selective activation of H2O2 production by exercise in the collateral-dependent arterioles. Because H2O2 is a stable and cell permeable molecule, the elevation of H2O2 detected in EX CLX arteriolar wall in response to adenosine stimulation (Figure 5) supports its role for vascular relaxation. Unfortunately, the endothelial source of H2O2 cannot be directly demonstrated in the present experimental settings. Although denudation has been considered, this approach not only eliminates the effector response (ie, production of superoxide from eNOS and the subsequent formation of H2O2) but also removes the sensor (ie, the endothelium) for agonist stimulation. Another limitation of the current study is the inability to quantify the produced H2O2 because current technology has not been advanced yet to allow possible quantitative measurement of H2O2 in the intact arteriolar wall. However, because H2O2 can contribute to about 10% to 40% of adenosine- and ionomycin-induced dilation in EX LCX arterioles (Figure 3C and 3D), we calculated from the H2O2 dose–response curve (supplemental Figure II) that about 3 to 10 ␮mol/L of extracellular H2O2 is required to elicit this range of dilation. Based on the permeability coefficient and the degradation kinetics for H2O2,11 the intracellular level of H2O2 was estimated to be 0.3 to 1.0 ␮mol/L. These levels are within the physiological range suggested in various mammalian cells.11 Adenosine-induced NO production was decreased in LCX arterioles (compared with nonoccluded LAD arterioles) from SED animals, providing supportive evidence for the potential role of impaired NO production in arterioles distal to coronary occlusion. Because L-NAME did not have effect on adenosine-induced dilation in SED LCX arterioles (Figure 2C), the slight increase (⬇40% above basal level) in adenosine-stimulated NO production appears to reflect nonvasoactive levels of NO production (ie, below the threshold for vasodilation) or, alternatively, may be attributed to the different experimental conditions of pressurized vessels (functional study) versus nonpressurized vessels (biochemical study). In exercise-trained animals, there was no difference in vasodilatory responses to adenosine between arterioles from nonoccluded LAD and occluded LCX region (Figure 1C). However, adenosine-stimulated NO production was not fully restored in LCX arterioles and L-NAME only

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slightly inhibited adenosine-induced dilation (Figure 2C). These results suggest that additional mediators, beside NO, may be involved in training-induced enhancement of vasodilation of these vessels. Indeed, the findings on the elevation of H2O2 production and the involvement of this molecule in vasodilation to eNOS activation in EX LCX arterioles (Figure 3C and 3D) explain the discrepancy between the functional data and NO assay. In sedentary pigs, the dilation of LCX arterioles to adenosine and ionomycin was improved by L-arginine supplementation. Because the effect of L-arginine was sensitive to L-NAME (Figure 4C and 4D), the involvement of limited substrate for NO synthesis during eNOS activation is evident in this regard. Interestingly, the NO production and vasodilation improved by L-arginine remained less than the responses of nonoccluded SED LAD arterioles. It is speculated that this remaining deficit is attributable to the reduction of eNOS mRNA levels as shown in a previous study using collateral-dependent arterioles from sedentary animals.2 Benefits of exercise on the cardiovascular system have long been recognized. The present study indicates that training-induced restoration of impaired vasodilation in collateral-dependent coronary arterioles involves contributions of both NO and H2O2. Although the endogenous role of H2O2 in the cardiovascular system remains obscure, accumulating evidence suggests that H2O2 plays a critical role in vasoregulation under physiological and pathophysiological conditions. For example, H2O2 may act as an EDHF in some vascular beds4 – 6 and may mimic reactive hyperemia in the skeletal muscle microcirculation.45 Furthermore, a recent in vivo study suggested that H2O2 plays an important role in autoregulation of coronary blood flow.46 In patients with coronary artery disease, increased H2O2 production may also play a critical compensatory role for impaired NO-mediated dilation in the ischemic coronary microcirculation.4 Because L-arginine supplementation enhances the endotheliumdependent NO-mediated dilation of coronary arterioles from collateral-dependent region in sedentary animals, it is tempting to suggest that the combination of L-arginine supplementation in concert with exercise-based programs25 may be beneficial in terms of improving coronary microvascular function and optimizing blood flow to collateralized myocardium distal to coronary occlusion.

Sources of Funding This work was supported by National Heart, Lung, and Blood Institute Grants HL-71761 (to L.K.) and HL-64931 (to J.P.).

Disclosures None.

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Arterioscler Thromb Vasc Biol.

April 2007

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Supplemental Data

Figure I. Vasodilation of coronary arterioles from nonoccluded (LAD) and collateral-dependent (LCX) regions of sedentary (SED) and exercise-trained (EX) animals in response to increasing concentrations of pinacidil (A) and sodium nitroprusside (B). Vasodilation to pinacidil and sodium nitroprusside was not different between groups. Number of vessel studied is indicated in parenthesis. 1

Supplemental Data

Figure II

Figure II. Vasodilation of coronary arterioles from nonoccluded (LAD) and collateral-dependent (LCX) regions of exercise-trained (EX) animals in response to exogenous H2O2. Vasodilation to H2O2 was not different between groups. H2O2-induced dilations were inhibited by catalase. Number of vessel studied is indicated in parenthesis.

2

Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion: Role of Hydrogen Peroxide Naris Thengchaisri, Robert Shipley, Yi Ren, Janet Parker and Lih Kuo Arterioscler Thromb Vasc Biol. 2007;27:791-798; originally published online January 18, 2007; doi: 10.1161/01.ATV.0000258416.47953.9a Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2007 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

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