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Circulation Research. 2003;92:e31-e40
Published online before print December 26, 2002, doi: 10.1161/01.RES.0000054200.44505.AB
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(Circulation Research. 2003;92:e31.)
© 2003 American Heart Association, Inc.

UltraRapid Communication

Role for Hydrogen Peroxide in Flow-Induced Dilation of Human Coronary Arterioles

Hiroto Miura, John J. Bosnjak, Gang Ning, Takashi Saito, Mamoru Miura, David D. Gutterman

From the Department of Veterans Affairs Medical Center, and Department of Medicine and Cardiovascular Research Center (H.M., J.J.B., D.D.G.), Milwaukee, Wis; Department of Microbiology and Molecular Genetics (G.N.), Medical College of Wisconsin, Milwaukee, Wis; and the 2nd Department of Internal Medicine (T.S., M.M.), Akita University, Akita City, Japan.

Correspondence to Hiroto Miura, Dept of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail hmiura{at}mcw.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Flow-induced dilation (FID) is dependent largely on hyperpolarization of vascular smooth muscle cells (VSMCs) in human coronary arterioles (HCA) from patients with coronary disease. Animal studies show that shear stress induces endothelial generation of hydrogen peroxide (H2O2), which is proposed as an endothelium-derived hyperpolarizing factor (EDHF). We tested the hypothesis that H2O2 contributes to FID in HCA. Arterioles (135±7 µm, n=71) were dissected from human right atrial appendages at the time of cardiac surgery and cannulated with glass micropipettes. Changes in internal diameter and membrane potential of VSMCs to shear stress, H2O2, or to papaverine were recorded with videomicroscopy. In some vessels, endothelial H2O2 generation to shear stress was monitored directly using confocal microscopy with 2',7'-dichlorofluorescin diacetate (DCFH) or using electron microscopy with cerium chloride. Catalase inhibited FID (%max dilation; 66±8 versus 25±7%; P<0.05, n=6), whereas dilation to papaverine was unchanged. Shear stress immediately increased DCFH fluorescence in the endothelial cell layer, whereas treatment with catalase abolished the increase in fluorescence. Electron microscopy with cerium chloride revealed shear stress–induced increase in cerium deposition in intimal area surrounding endothelial cells. Exogenous H2O2 dilated (%max dilation; 97±1%, ED50; 3.0±0.7x10-5 mol/L) and hyperpolarized HCA. Dilation to H2O2 was reduced by catalase, 40 mmol/L KCl, or charybdotoxin plus apamin, whereas endothelial denudation, deferoxamine, 1H-1,2,4-oxadiazole-[4,3-a]quinoxalin-1-one, or glibenclamide had no effect. These data provide evidence that shear stress induces endothelial release of H2O2 and are consistent with the idea that H2O2 is an EDHF that contributes to FID in HCA from patients with heart disease. The full text of this article is available at http://www.circresaha.org.


Key Words: human • coronary microcirculation • flow-induced dilation • hydrogen peroxides


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Physiologically, shear stress plays a critical role in the regulation of vascular tonus and vascular homeostasis, contributing to the maintenance of tissue perfusion and vascular integrity. Shear stress–induced release of nitric oxide (NO) from endothelial cells is widely recognized as one of the most important and common mechanisms for shear-induced vasomotion. For example, flow-induced release of NO is responsible for the mediation of flow-induced vasodilation (FID).1–3

Animal studies have reported that the contribution of NO to FID is reduced as oxidative stress increases in the presence of risk factors for cardiovascular disease such as hypercholesterolemia4 and hypertension.5 In humans, in vivo and in vitro studies have demonstrated that relaxant factor(s) other than NO compensate to maintain FID when NO availability is reduced.6,7 We recently reported that FID is mediated largely by endothelium-derived hyperpolarizing factor (EDHF) in human coronary arterioles (HCAs) isolated from patients undergoing cardiac surgery3; however, the chemical nature of the specific EDHF remains unknown. Hydrogen peroxide (H2O2) was first proposed as an EDHF by Matoba et al.8 These investigators showed that catalase, a H2O2 scavenger inhibits vasodilation and hyperpolarization to acetylcholine (ACh) in mouse mesenteric arteries.8 Pathological generation of reactive oxygen species (ROS) including H2O2 has been described in diseased vessels under static conditions.9,10 Furthermore, an animal study indicates that the marked generation of endothelium-derived H2O2 contributes to agonist-induced vasodilation in disease states.11

The purpose of this study was to investigate in HCA from patients with heart disease, whether H2O2 is an EDHF, whether it contributes to FID, and whether shear stress elicits endothelial H2O2 generation and release. We also determined the pharmacological characteristics of the vascular response to H2O2, because EDHF is generally recognized to induce membrane hyperpolarization and vasodilation through opening Ca2+-activated K+ channels (KCa).3,12,13


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
HCAs were prepared as reported previously.3,13–15 Briefly, HCA were dissected from fresh specimens of right atrial appendage obtained from patients undergoing cardiopulmonary bypass procedures as discarded surgical specimens. Procedures for harvesting tissue samples were in accordance with guidelines established by the local Institutional Review Boards. Demographic data and diagnoses were obtained from hospital records and recorded at the time of surgery.

Videomicroscopy
Videomicroscopy was performed as reported previously.3,13–15 Briefly, isolated HCAs were transferred to a organ chamber containing Krebs solution of the following composition (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 20, Na2EDTA 0.026, and glucose 11, pH 7.4, cannulated with glass micropipettes (30 to 50 µm internal diameter, matched for impedance; see next section) and secured. All side branches were tied off with cotton threads. The preparation was transferred to the stage of an inverted microscope (CK2, Olympus) coupled to a CCD camera (WV-BL200, Panasonic) and video micrometer (VIA-100K, Boeckeler Instruments, Inc). Pharmacological agents other than catalase were added to the oxygenated external bathing solution (37°C).

The vasomotor and endothelial function was confirmed by examining constriction to 50 mmol/L KCl and dilation to bradykinin (BK, 10-7 mol/L), respectively.13 Because HCAs develop varying degrees of spontaneous myogenic tone (10% to 60%), ACh (5x10-8 to 5x10-7 mol/L), a potent and stable vasoconstrictor of HCA,14–16 was, if needed, added to adjust tone to a level between 30% to 60% of passive diameter so that all vessels were constricted to a similar level.

Generation of Intraluminal Flow
Both proximal (inflow) and distal (outflow) micropipettes were connected with silicone tubing to a pressure-servo syringe system (Living Systems Inc), which was used to control intraluminal flow without changing intraluminal pressure.2,17,18 The pipettes had similar dimensions and equivalent resistances to flow, as assessed by the changes in perfusion pressure in response to increments of flow by a calibrated flow control peristaltic pump (model FC, Living Systems Inc). Pulse dampers were used to minimize oscillations in flow caused by the pump. Intraluminal flow was monitored with a flowmeter (model 4552, Gilmont Instruments, Inc).3 Shear stress was calculated by using the flowing formula: 4{eta}Q/{pi}r3, where {eta} is viscosity of the perfusate (0.007 poise at 37°C), Q is perfusates flow (mL/sec), and r is vessel radius (cm).

Vessels were initially pressurized at 60 mm Hg without flow to confirm absence of leaks either at the site of cannulation or via undetected side-branches. Vessel preparations with leaks (flow>0) were discarded ({approx}5% of experiments).

Experimental Protocols of Flow-Induced Vasodilation
Vessel diameter was examined at different flow rates (1, 3, 5, 10, 15, 20, and 25 µL/min). After determination of the control relationship between vessel internal diameter and flow rate, vascular responses to flow were reexamined in the presence of catalase (1000 U/mL), inactivated catalase,19 or the combination of catalase and superoxide dismutase (SOD, 200 U/mL), both of which were applied intraluminally and extraluminally. Maximal vasodilator capacity was determined by addition of papaverine (an endothelium-independent dilator, 10-4 mol/L). In some experiments, the vasodilation to papaverine (10-7 to 10-4 mol/L) was examined in the absence or presence of catalase.

H2O2-Induced Vasodilation
To determine the mechanism for vasodilation to H2O2 on HCA, vascular responses to exogenous H2O2 (10-7 to 3x10-4 mol/L) were examined in the absence or presence of catalase (1000U/mL), inactivated catalase,19 deferoxamine (10-3 mol/L, an inhibitor of the formation of the hydroxyl radical, a ROS derived from H2O2), or 1H-[1,2,4]-oxadiazole-[4,3-a]quinoxalin-1-one (ODQ, 5x10-6 mol/L, a selective inhibitor of guanylate cyclase).15,20 In some experiments, endothelium-denuded HCAs13 were used to determine the role of endothelial cells in vascular response to H2O2.

To examine whether membrane hyperpolarization contributes to H2O2-induced changes in vessel diameter, KCl (40 mmol/L) was used to nonspecifically block K+ channels in HCAs. In separate studies, the effects of the combination of charybdotoxin (CTX, 10-7 mol/L, a selective blocker of large and intermediate conductance KCa) and apamin (10-6 mol/L, a selective blocker of small conductance KCa), or glibenclamide (10-6 mol/L, a selective blocker of ATP-sensitive K+ channels [KATP]) were also tested. In these protocols, all chemicals were applied extraluminally.

At the end of experiments, maximal passive diameters were obtained by incubating vessels with Ca2+-free Krebs solution in the presence of papaverine (10-4 mol/L).

Measurement of Vascular Smooth Muscle Membrane Potential
Resting membrane potential (Em) of vascular smooth muscle cells (VSMCs) and changes in Em to endothelin-1 and H2O2 were measured as described previously.3,13,21 Briefly, HCAs were cannulated, pressurized, and suspended in a 20-mL tissue bath. Em was measured by impaling the vessel from the adventitial surface with a glass microelectrode filled with 3 mol/L KCl and connected to a high-impedance biological amplifier (Axoclamp, Axon Instruments).

Detection of Endothelial H2O2 Production With Confocal Microscopy
A closed imaging chamber (model RC-20, Warner Instrument Corp) was used for detection of fluorescence in HCAs using confocal microscopy. HCAs were placed in the chamber and filled with HEPES buffer (pH7.4 at 37°C). One end of the vessel was cannulated with a micropipette (internal diameter; 30 to 40 µm) inserted to the perfusion inlet of the chamber and secured, and the other end of the vessel was left opened. The chamber was attached onto a heater platform (model PH-5, Warner Instrument Corp) connected to a heater controller chamber system (model TC-344B, Warner Instrument Corp) to keep the system at 37°C and mounted inversely onto the stage of a confocal microscope. After 60-minute equilibration, HCAs were loaded for 30 minutes with a fluorescence probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH; 5x10-6 mol/L, Molecular Probes),22,23 by superfusing HEPES buffer containing DCFH and N{omega}-nitro-L-arginine (10-4 mol/L, an NO synthase inhibitor) in the absence or presence of catalase (1000U/mL).24 After loading the probe, HCAs were perfused to generate shear stress ({cong}21±2 dyn/cm2) by switching the perfusion from a secondary inlet port to a micropipette cannulating the vessel, and fluorescence images were obtained during 30-minute exposure to shear stress. In some experiments, fluorescence images were obtained without intraluminal flow for 30 minutes as a time control. Fluorescence was excited by 488-nm line of a krypton-argon laser, and emission at 505 to 535 nm was recorded. Images were obtained with a Bio-Rad MRC 600 laser scanning confocal imaging system mounted on a Nikon Optiphot microscope using x60 or x100 oil-immersion objective lens. Images were analyzed on a computer with the software program MetaMorph (Universal Imaging Corp).

Detection of Extracellular H2O2 Release With Electron Microscopy
To estimate and localize shear stress–induced H2O2 production, histochemical electron microscopy was performed with cerium chloride,25,26 a staining method that allows the specific visualization of H2O2 production in tissues. Briefly, three vessels were isolated from each subject and incubated in 10 mmol/L Tris-maleate saline buffer (pH 7.4) containing 5.5 mmol/L glucose, 7% sucrose, and 3-amino-1H-1,2,4-triazole (10-2 mol/L) for 30 minutes. Two of three vessels were cannulated with a glass pipette and placed in the bath filled with the buffer containing cerium chloride (10-3 mol/L). Vessels were exposed to shear stress ({approx}20 dyn/cm2) by perfusing the buffer with cerium chloride for 2 minutes in the presence or absence of catalase (1000U/mL). One of three vessels was treated with the buffer containing cerium chloride for 2 minutes without perfusion. Subsequently, vessels were then rinsed with the buffer to wash out unreacted cerium ions, fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH7.4, rinsed and postfixed with 1% OsO4, and then dehydrated and embedded in epoxy resin. Thin cross sections of each vessel were obtained with an UltraCut E microtome. Sections were stained with uranyl acetate and lead citrate and examined with a Hitachi 600 transmission electron microscope at 75 kV.

Materials
CTX was obtained from Research Biochemicals international, DCFH came from Molecular Probes, and all other chemicals were from Sigma Chemical Co. Glibenclamide was prepared in dimethyl sulfoxide and diluted in saline with 1.0 N NaOH, and pH was adjusted with 0.1 N HCl to 7.4. DCFH and ODQ were prepared in dimethyl sulfoxide. All others were dissolved in distilled water. All concentrations represent the final molar concentrations (mol/L) in the organ chambers.

Statistical Analyses
FID and agonist-induced dilation are expressed as a percent, with 100% dilation representing the change from the constricted diameter to the maximal diameter obtained by addition of papaverine (10-4 mol/L). Maximal passive diameters were used as the maximal diameter for papaverine-induced responses. DCFH fluorescence intensity was normalized as a percent change in intensity levels from baseline (100%). Statistical comparisons of maximal percent vasodilation, ED50 values (the molar concentration of dilator that produced a 50% maximal response), Em values, and change in fluorescence intensities under different treatments were performed using paired or unpaired Student’s t test, whenever applicable. A two-factor repeated measures analysis of variance was used to compare dose (shear)/response (factor 1) relationships between the treatment groups (factor 2). Interactions were noted between dose (shear) and treatments groups. Corollary dose (shear)–specific contrasts were tested using Bonferroni adjusted t test whenever the interactions were statistically significant. Multivariate analysis was performed, and regression models were made as previously described.13 All procedures were performed using "proc mixed" and "proc reg" programs of SAS for Windows, version 8. Statistical significance was defined as P<0.05. All data are described as mean±SEM. For all data, n indicates the number of patients.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Seventy-one HCAs with a mean maximal passive internal diameter of 135±7 µm (range 67 to 298 µm) were used. Patient demographics including diagnoses are summarized in the Table.


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  		Table 1. Demographics (n=71)

Figure 1 shows the effect of catalase on FID and papaverine responses. An increase in flow produced potent vasodilation, which was attenuated by catalase (Figure 1A; %max. dilation, 21±9 versus control 60±9% at 25 µL/min; P<0.05, n=6). Figure 1B shows the relationship between calculated shear stress and FID. FID closely correlated to shear stress. Catalase decreased vasodilation in response to shear stress. In contrast, vasodilation to papaverine was unchanged in the presence of catalase (Figure 1C; %max dilation, 91±4% versus control 91±5%; -log[ED50], 5.2±0.1 versus control 5.8±0.6; P=NS, n=4, respectively). Catalase inactivated by heating19 also had no inhibitory effect on FID (%max dilation, 62±10% versus control 58±8%; P=NS, n=3). The combination of catalase and SOD also reduced FID (%max dilation, 10±6% versus control 50±13% at 25 µL/min; P<0.05, n=4). These results indicate that the inhibitory effect of catalase on FID is unlikely to be nonspecific, suggesting an important role for H2O2 in mediating FID in HCAs.



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  		Figure 1. Role of endogenous H2O2 in FID in HCAs. A, Catalase (1000 U/mL) attenuated FID (#P<0.05, n=6). B, FID was closely correlated to calculated changes in shear stress. C, Vasodilation to papaverine was unchanged by catalase (P=NS, n=4), indicating specificity.

We evaluated endothelial H2O2 generation in response to shear stress in HCAs using confocal microscopy with DCFH, an H2O2-sensitive fluorescence probe.22,23 Figure 2, left, shows representative images obtained from a vessel with confocal microscopy. In those images, endothelial cells (EC) are recognized as cells positioned parallel to the vessel axis, and VSMCs (SM) are observed as cells oriented perpendicular to the vessel axis. Intraluminal flow produced a marked increase in fluorescence in endothelial cells and minimal increase in VSMCs (Figures 2a through 2d). In vessels treated with catalase, the fluorescence intensities in endothelial cells and VSMCs were unaltered after exposure to intraluminal flow (Figures 2e through 2h). Summary data showing the change in endothelial fluorescence intensity to shear stress is shown in Figure 2, right. After initiating intraluminal flow, the fluorescent intensity in endothelial cells was significantly increased within a minute (138±9%; P<0.05 versus baseline [100%] at 5 minutes). Catalase completely abolished the increase in fluorescence to flow (93±3%; P<0.05 versus baseline [100%] and in the absence of catalase at 5 minutes, respectively). The fluorescence intensity increased only by 9% (P<0.05 versus endothelial cells at 5 minutes). We confirmed that endothelial denudation abolishes shear-induced increase in DCFH fluorescence by obtaining fluorescent images of vessels (n=3, data not shown). Time controls showed no changes in fluorescence during the protocol (data not shown, n=3). These results indicate that shear stress immediately elicits time-dependent production of H2O2 in endothelial cells of HCA.



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  		Figure 2. Shear stress–induced H2O2 generation in HCA. Left, Representative images of DCFH fluorescence in an HCA before (a and e) and after exposure to shear stress (b through d and f through h) in the absence or presence of catalase (1000 U/mL). a through d, Shear stress induces an increase in fluorescence intensity in the endothelial cell layer (EC) but not in the VSMC layer (SM). e through h, Increase in fluorescence to shear stress was not observed in vessels treated with catalase. Right, Summary data shows that shear stress induced endothelial H2O2 production occurred in a time-dependent manner (*P<0.05 vs baseline, n=5). Treatment with catalase completely abolished H2O2 production ({dagger}P<0.05 vs control, n=4).

To more directly determine the role of H2O2 in FID, electron microscopy was conducted to detect cerium deposition, which is produced by the reaction of H2O2 with cerium ions,25,26 in layers of vessels exposed to shear stress. In vessels without exposure to shear stress, few cerium depositions were observed in the intima (Figures 3A and 3D). In contrast, exposure to shear stress markedly increased cerium depositions in the intimal layer, especially surrounding endothelial cells (Figures 3B and 3E), while treatment with catalase inhibited the production of cerium depositions (Figures 3C and 3F). Figure 3H shows whole vascular wall of vessels exposed to shear stress in the absence of catalase. Cerium depositions were seen throughout the vascular wall with the majority of cerium deposits in the intimal layer compared with the medial layer and most occurring adjacent to membrane structures. In contrast, few depositions were observed in the vascular walls of vessels without shear stress (Figure 3G) or those vessels treated with catalase (Figure 3I). These findings suggest that endothelial cells release H2O2 in response to shear stress and this H2O2 might migrate to interact with VSMCs.



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  		Figure 3. Representative transmission electron microscopic images of HCAS treated with cerium chloride to show localization of H2O2 generated in response to shear stress. A and D, Control incubation of HCAS without flow produces few cerium depositions in the intimal layer. B and E, Exposure of HCA to shear stress yields numerous cerium depositions (arrow) in the intimal layer, especially around perisarcolemmal portions of the endothelial cells. C and F, Treatment with catalase markedly inhibits shear-induced generation of H2O2 in the intima. H, Cerium depositions are seen throughout the vessel wall in vessels exposed to shear stress compared with the vessels without shear stress (G) or treated with catalase (I). Depositions exist in the intima more than in the media. Magnifications, x10000 (A through C) and x2500 (G through I). Scale bars=1 µm (A through I). L indicates lumen; I, intimal layer; M, medial layer; E, endothelial cell; and V, VSMC.

To determine the mechanism of H2O2-mediated vasodilation, the vascular response to exogenous H2O2 was examined in HCAs. Because H2O2 can be converted to hydroxyl radical via the Haber-Weiss reaction, H2O2-induced vasodilation may be elicited directly by H2O2 or indirectly by hydroxyl radicals.27 The effect of scavenging H2O2 by catalase or inhibiting the formation of hydroxyl radicals by deferoxamine was tested. The presence of catalase inhibited vasodilation to H2O2 (Figure 4A; max dilation, 22±3% versus control 97±1%; P<0.05, n=4), whereas deferoxamine (%max dilation, 98±1% versus control 99±1%; -log[ED50]; 5.0±0.3 versus control 5.1±0.2; P=NS, n=4) or inactivated catalase19 (data not shown) had no effect on vasodilation to H2O2.



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  		Figure 4. H2O2-induced dilation of HCAs. A, Catalase abolished H2O2–induced dilation of HCAs (*P<0.05, n=4). B, Vasodilation to H2O2 was unaltered by endothelial denudation (P=NS, n=4). C, High concentration of KCl (40 mmol/L) attenuated vasodilation to H2O2. (*P<0.05 vs control, n=6). D, After membrane depolarization and vasoconstriction with endothelin-1 (ET), H2O2 induced a dose-dependent membrane hyperpolarization and vasodilation (*P<0.05 vs ET, n=5).

H2O2 may act on endothelial cells to elicit vasodilation.28 However, endothelial denudation had no effect on vasodilation to H2O2 (Figure 4B; %max dilation, 97±2% versus control 98±1%; -log[ED50], 4.8±0.3 versus control 4.8±0.3; P=NS, n=4). Guanylate cyclase may also mediate vasodilation to H2O2.29 Vasodilation to H2O2 was unchanged by ODQ, an inhibitor of guanylate cyclase (%max dilation, 95±2% versus control 97±1%; -log[ED50], 4.8±0.3 versus control 5.1±0.2; P=NS, n=4). These results suggest that H2O2–induced vasodilation is mediated by endothelium-independent mechanisms, which do not involve guanylate cyclase activation or hydroxyl radical formation in HCAs.

It has been reported that membrane hyperpolarization through K+ channel activation contributes largely to vasodilation not only to H2O2 but also to shear stress.3,8,30 Consistent with these observations, a high concentration of KCl (40 mmol/L) reduced H2O2-induced vasodilation (Figure 4C; %max dilation, 64±5% versus control 100±0%; -log[ED50], 4.1±0.1 versus control 4.8±0.2; n=6, P<0.05 for both comparisons). To directly support the idea that vasodilation to H2O2 is dependent largely on membrane hyperpolarization, changes in Em of VSMCs were measured. As shown in Figure 4D, H2O2 produced simultaneous vasodilation and membrane hyperpolarization in a dose-dependent manner in HCA. This is consistent with our previous report that FID is dependent on membrane hyperpolarization via K+ channel activation in the human coronary microcirculation.3

We next examined which type of K+ channel(s) mediates vasodilation to moderate doses of H2O2, because the contribution of endogenously generated H2O2 to FID is approximately 40%. Glibenclamide did not alter H2O2-induced vasodilation in HCAs (Figure 5A; %max dilation, 35±18% versus control 49±15% at 10-5 mol/L; P=NS, n=6), whereas the combination of CTX and apamin significantly decreased the dilation (Figure 5B; %max dilation, 3±2% versus control 49±14% at 10-5 mol/L; P<0.05, n=6). These results suggest that KCa plays a role in mediating H2O2-induced dilation of HCAs.



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  		Figure 5. Effect of K+ channel blockers on H2O2-induced vasodilation in HCA. A, Vasodilation to H2O2 was unchanged in the presence of glibenclamide (10-6 mol/L) (P=NS, n=4). B, Combination of CTX (10-7 mol/L) and apamin (10-6 mol/L) significantly reduced H2O2–induced dilation ({dagger}P<0.01, *P<0.05 vs control, n=6).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
This study is the first to directly examine H2O2 as a potential mediator of FID in HCA. The major new findings are 4-fold. First, H2O2 contributes to FID in HCAs. Second, shear stress elicits endothelial generation and release of H2O2. Third, H2O2-induced vasodilation is not endothelium-dependent but is mediated by direct effects on the underlying VSMCs in HCA. Fourth, vasodilation to H2O2 occurs as a result of membrane hyperpolarization consequent to the opening of KCa in VSMCs. Taken together, these findings suggest that endothelium-derived H2O2 plays an important role in FID in HCA from patients with heart disease. H2O2 is most likely an EDHF in the human coronary microcirculation.

Flow-Induced Vasodilation
The vasomotor response to shear stress is varied among species and vasculatures. Endothelium-dependent vasodilator responses to shear stress have been demonstrated in a variety of vessels including rat cremaster and gracilis arterioles, and porcine coronary arterioles,1,2,31 whereas endothelium-independent vasoconstriction is observed in cat and rat cerebral arteries.32,33 We previously reported that shear stress elicits endothelium-dependent vasodilation in HCA.3 In the present study, we show that H2O2 contributes to the FID in HCA.

Shear Stress Elicits Endothelial Production and Release of H2O2
Numerous in-vivo and in vitro studies have demonstrated that shear stress stimulates the production and release of ROS from endothelial cells, most notably superoxide.23,34,35 Human endothelial cells also generate superoxide in response to shear stress.35 Superoxide is rapidly dismutated to H2O2. This may account for the finding by Hsieh et al23 of shear stress-induced H2O2 production in cultured human umbilical vein endothelial cells. In contrast to the pathological function of H2O2,9,10 a physiological role in vasodilation has been demonstrated. For example, vasorelaxation to the calcium ionophore A23187 in aortas of spontaneously hypertensive rats largely involves H2O2.11 In the present study, we demonstrated that endothelial H2O2 generation is associated with vasodilation to shear stress, a key physiological stimulus for dilation.

Scavenging extracellular H2O2 with catalase could reduce vasodilation to agonists such as BK and ACh in the vasculatures among different species.8,36–38 Interestingly, it has been reported that SOD and catalase can reduce vasoconstriction to flow in rat and cat cerebral arteries.32,33 Laurindo et al34 have reported that shear stress induces endothelial superoxide generation with increases in the radical product in plasma of perfused rabbit vessels. These studies support our finding with electron microscopy that endothelial cells release H2O2 extracellularly in response to shear stress. The present study is the first demonstration that H2O2 is involved in FID, indicating H2O2 as an endothelium-derived and transferable vasodilator in FID.

Vasoreactivity to H2O2
H2O2 induces vasorelaxation in an endothelium-dependent manner in rabbit aorta28. However, we found that H2O2-induced dilation is endothelium-independent in HCAs, consistent with other studies in porcine coronary arteries.30

H2O2 may be converted to hydroxyl radicals via Haber-Weiss reaction and this may be necessary for dilation of cat cerebral arteries.27 However, it is not critical in H2O2–induced dilation of porcine coronary arteries39 or mouse mesenteric arteries.8 In the present study, deferoxamine had no effect on vasodilation of HCA to H2O2

It has been also reported that H2O2-induced vasodilation is mediated by KCa in porcine coronary arteries,30,39 rat cerebral arteries,37 and mouse mesenteric arteries,8 by KATP in cat cerebral arterioles,40 or by soluble guanylate cyclase in bovine pulmonary arteries.29 Electrophysiological investigations have also revealed H2O2-induced direct activation of KCa in VSMCs isolated from coronary arteries.30,39 In the present study, H2O2 produced membrane hyperpolarization and vasodilation of HCA, which was sensitive to CTX and apamin but resistant to glibenclamide, deferoxamine, and ODQ. These findings suggest that in HCAs, H2O2 elicits membrane hyperpolarization and vasodilation through the activation of KCa but not through KATP, hydroxyl radical formation, or soluble guanylate cyclase.

H2O2 as an EDHF
Although FID is mediated largely or in part by NO, prostaglandins, or both in most vessels1,2,31 including HCAs isolated from patients without coronary artery disease (CAD),3 EDHF contributes largely to FID in HCA from patients with heart disease,3 porcine epicardial coronary arteries,41 and rat mesenteric arteries.42 Animal studies have demonstrated that endothelium-derived H2O2, which is first proposed as an EDHF by Matoba et al8 compensates for loss of NO activity and contributes largely to endothelium-dependent vasodilation.43 These studies are consistent with the suggestion of the present study that H2O2 acts as an EDHF and maintains FID in HCA isolated from patients with heart disease. The mechanism of dilation to H2O2 involves hyperpolarization and opening of KCa. Therefore, H2O2 is likely an EDHF involved in FID in the human coronary microcirculation.

Potential Limitations of the Study
The local extracellular concentration of H2O2 generated in response to shear stress is not known. In the present study, approximately 10-5 mol/L of exogenous H2O2 was required to induce a catalase-sensitive vasodilation that was of similar magnitude to that produced by shear stress (max dilation {cong}40%). ED50 of exogenous H2O2-induced dilation was approximately 3x10-5 mol/L in HCAs, whereas other studies reported ED50 of 10-5 to 10-4 mol/L in mouse small mesenteric arteries8 and 2.5x10-4 mol/L in porcine coronary arteries.30 The different sensitivity of vessels to H2O2 may be dependent on vessel size.40 Consentino et al11 reported in aortas from spontaneously hypertensive rats that endogenously generated H2O2 at 10-8 to 10-7 molar ranges contributes largely to vasorelaxation to A23187. Therefore, the concentration of H2O2 required to induce vasodilation to physiological stimuli such as shear stress, ACh,8 and BK37 may be lower than that required for dilation to exogenous H2O2. This could be explained by the interaction of H2O2 with endogenous catalase, by other redox reactions that inactivate H2O2 or convert it to other radical species before diffusing to the active site, or by the additive effect of H2O2 on other vasorelaxant mechanisms.

Although there are several enzymatic sources of ROS in endothelial cells including eNOS, cyclooxygenase, cytochrome P450, NADH oxidase, xanthine oxidase, and sites along the mitochondrial respiratory chain, the source of radicals underlying shear stress–induced ROS production remains unclear. eNOS and cyclooxygenase are activated by shear stress,2,31,44 but activation of these enzymes is not associated with ROS production during shear stress.33,34 De Keulenaer et al reported that shear stress stimulates superoxide generation from NADH oxidase.35 It is, however unlikely that all H2O2 involved in FID originates intracellularly, because intracellular antioxidant systems including catalase, glutathione peroxide/reductase, and others scavenge intracellular H2O245 and because the relatively cell-impermeable catalase inhibited the increase in DCFH fluorescence to shear stress in the present study and others.23 Thus, we presume that most H2O2-inducing FID may be derived from membrane-bound enzymes or cytosolic enzymes adjacent to the membrane.

A portion of the dilation to flow was not blocked by catalase. Because the dose of catalase used was sufficient to completely block responses to pharmacological doses of H2O2, it is likely that mechanisms not involving H2O2 also contribute to FID in HCAs. In this regard, we have previously shown that arachidonic acid metabolites of cytochrome P450, possibly epoxyeicosatrienoic acids (EETs), are important in FID under the conditions of inhibiting NO syntheses and cyclooxygenase.3 L-Arginine analogues increase ROS production from "normal" eNOS, while they decrease ROS from "dysfunctional" eNOS,46 which is proposed a source of H2O2.8 ROS could affect arachidonic acid metabolism of cytochrome P450.47,48 Therefore, the multiple contribution of more than one EDHF (H2O2 and EETs) or the interaction between EDHFs should be considered. Thus, the inhibitory effect of catalase on FID in our study might be attributed to an alteration in EET formation. Alternatively, superoxide and subsequently H2O2 can be produced by cytochrome P450.24 It is possible that cytochrome P450 may be a source of either EETs, H2O2, or both to induce FID in HCA.

It is possible that H2O2 contributes less to FID in vivo, because greater scavenging of ROS may occur via antioxidants in blood cells and plasma.34 However, in vivo studies have demonstrated that vasodilation of rat and cat cerebral arteries to BK is abolished by catalase, and that vasodilation to H2O2 itself occurs even at lower concentrations than used in the present study, suggesting a similar role for H2O2 in vivo.36,37,40

Clinical Implications
A previous study from our laboratory showed a more prominent role of EDHF in mediating FID in patients with CAD compared with subjects without CAD,3 indicating that EDHF, including H2O2, plays a critical role in compensation for impairment in NO-mediated vasodilation,3,43 consistent with a regulatory role of H2O2 in the coronary microcirculation designed to maintain myocardial perfusion in chronic disease states. We could not test the role of H2O2 in FID in "normal" HCAs. When patients are divided into two groups, patients with or without CAD, H2O2 contributes to FID in vessels from patients with CAD (67% of the dilation) more than those from patients without CAD (44% of the dilation). This is consistent with the finding showing a prominent role of H2O2 in mediating endothelium-dependent relaxation of aorta in diseased animal models.11 In addition, H2O2 mediates BK-induced vasodilation in mesenteric arteries from patients without CAD.38 Thus, we speculate that H2O2 may be an important physiological mediator of FID in HCAs, and that the presence of coronary risk factors or CAD may augment the contribution of H2O2 to FID.

H2O2 is a relatively stable reactive oxygen intermediate and contributes importantly to inflammation, ischemia/reperfusion injury, and atherosclerosis.49–51 It may, therefore have a pathophysiological as well as a normal signaling function. In chronic disease states such as hypercholesterolemia and diabetes mellitus, oxidative stress and H2O2 levels are increased due to the enhanced generation of superoxide from several sources, leading to vascular dysfunction.52 Endothelial H2O2 production to shear stress may be a double-edged sword in the human coronary microcirculation.

In summary, endothelium-derived H2O2 contributes to FID in isolated HCA from patients with heart disease. Vasodilation to H2O2 is associated with membrane hyperpolarization of VSMCs via KCa opening, suggesting H2O2 is an EDHF in the human coronary microcirculation. This dilator mechanism may play an important role in maintaining myocardial perfusion when levels are NO are reduced, but it may also lead to the enhanced development of vascular pathology including atherosclerosis.


*    Acknowledgments
 
This study was supported by a VA Merit Award and grants from NIH (HL65203 and HL62852).

Received November 6, 2002; revision received December 9, 2002; accepted December 13, 2002.


*    References
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