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(Circulation Research. 2003;93:573.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Mitochondrial Sources of H₂O₂ Generation Play a Key Role in Flow-Mediated Dilation in Human Coronary Resistance Arteries

Yanping Liu, Hongtao Zhao, Hongwei Li, B. Kalyanaraman, Alfred C. Nicolosi, David D. Gutterman

From the Departments of Internal Medicine (Y.L., H.L., D.D.G.), Biophysics (H.Z., B.K.), and Surgery (A.C.N.), Free Radical Research Center (H.Z., B.K.) and Cardiovascular Center (Y.L., H.L., D.D.G.), Medical College of Wisconsin; Zablocki VA Medical Center (Y.L., D.D.G.), Milwaukee, Wis.

Correspondence to Yanping Liu, MD, PhD, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ypliu{at}mcw.edu

	Abstract

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We previously showed that hydrogen peroxide (H₂O₂) contributes to flow-induced dilation in human coronary resistance arteries (HCRAs); however, the source of this H₂O₂ is not known. We hypothesized that the H₂O₂ is derived from superoxide (O₂^·-) generated by mitochondrial respiration. HCRAs were dissected from right atrial appendages obtained from patients during cardiac surgery and cannulated with micropipettes. H₂O₂-derived radicals and O₂^·- were detected by electron spin resonance (ESR) using BMPO as the spin trap and by histofluorescence using hydroethidine (HE, 5 µmol/L) and dichlorodihydrofluorescein (DCFH, 5 µmol/L). Diameter changes to increases in pressure gradients (20 and 100 cm H₂O) were examined in the absence and the presence of rotenone (1 µmol/L), myxothiazol (100 nmol/L), cyanide (1 µmol/L), mitochondrial complex I, III, and IV inhibitors, respectively, and apocynin (3 mmol/L), a NADPH oxidase inhibitor. At a pressure gradient of 100 cm H₂O, ubisemiquinone and hydroxyl radicals were detected from effluents of vessels. Including superoxide dismutase and catalase in the perfusate reduced the ESR signals. Relative ethidium and DCFH fluorescence intensities in HCRAs exposed to flow were enhanced (1.45±0.15 and 1.57±0.12, respectively compared with no-flow) and were inhibited by rotenone (0.87±0.17 and 0.95±0.07). Videomicroscopic studies showed that rotenone and myxothiazol blocked flow-induced dilation (% max. dilation at 100 cm H₂O: rotenone, 74±3% versus 3±13%; myxothiazol, 67±3% versus 28±4%; P<0.05). Neither cyanide nor apocynin altered flow-induced dilation. These results suggest that shear stress induced H₂O₂ formation, and flow-induced dilation is derived from O₂^·- originating from mitochondrial respiration.

Key Words: coronary • mitochondria • shear stress • free radicals

	Introduction

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Blood flow through an artery stimulates endothelial release of bioactive metabolites such as nitric oxide (NO)¹ and prostacyclin (PGI₂)² that mediate vasodilation. It has been suggested that shear is one of the most important physiological stimuli for vasodilation, and in large arteries may be critical for preventing atherosclerosis through release of the endothelial-derived antiproliferative nitric oxide.³

More recently laminar shear has been shown to induce changes in the vascular redox state. Flow-dependent release of superoxide (O₂^·-) is also observed in conduit arteries from normal animals.⁴ In cultured endothelial cells, O₂^·- is released during laminar or oscillatory shear.⁵ An increase in vascular production of reactive oxygen species (ROS) has been traditionally considered a pathological response that leads to impaired vasomotor function. This concept is based on the observation that excess O₂^·- generated in blood vessels by hypertension, hypercholesterolemia, or diabetes quenches NO produced by the endothelium and thereby reduces vasodilation. However the situation is more complex, especially in the microcirculation where NO participates less in vasomotor responses.⁶ Recent investigations show that ROS can play an important supportive role in regulating vascular function. For example, studies from our laboratory indicate that hydrogen peroxide (H₂O₂), a product dismutated from O₂^·-, hyperpolarizes and dilates human coronary arterioles through opening of Ca²⁺-activated K⁺ channels.⁷ Enhanced production of H₂O₂ is observed when the flow rate is increased in human coronary resistance vessels. Catalase, a scavenger of H₂O₂, greatly inhibited flow-induced dilation (FID).⁷ These observations suggest that H₂O₂ as an EDHF as suggested by others⁸ contributes to FID in human coronary arterioles.

However, there is paucity information about the source of H₂O₂ in FID. A variety of endogenous enzyme systems are known to generate superoxide including NOSIII, cyclooxygenase, NADPH oxidase, and xanthine oxidase. Initial observations suggested that inhibiting NOS and cyclooxygenase did not alter FID. In most cells, the majority of superoxide is generated from mitochondria via cellular respiration. Mitochondrial-derived O₂^·- is formed during ubisemiquinone autoxidation and is considered the stoichiometric precursor of H₂O₂.^9,10 The purpose of this study was to determine in human coronary resistance vessels from patient with heart disease, whether mitochondria are involved in shear-induced production of H₂O₂ that contributes to FID.

	Materials and Methods

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Preparation of Coronary Arteries
Human coronary resistance arteries (HCRAs) were derived from the right atrial appendage of subjects undergoing cardiopulmonary bypass surgery. Removed tissue was placed in oxygenated physiological salt solution (PSS) as described previously.¹¹ HCRAs were dissected from the endocardial surface of the appendage and prepared for videomicroscopy, histofluorescence, or electron spin resonance (ESR).

Electron Spin Resonance Measurements
Collecting Perfusate Effluent
HCRAs were cannulated on glass micropipettes in an organ chamber filled with PSS as described previously.¹² Each pipette was attached to an individual pressure reservoir. The PSS was warmed to 37°C, continuously circulated, and bubbled with 21% O₂, 5% CO₂, and 74% N₂. After 60 minutes of equilibration at an intraluminal pressure of 60 mm Hg, arteries were perfused with spin trap 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO, 25 mmol/L) at pressure gradient of 100 cm H₂O. Effluents were collected every minute (flow rate, 52±15 µL/min; shear stress, 18 to 25 dyn/cm²) from the outflow micropipette and immediately frozen with liquid nitrogen.

Free-Radical Measurements
Effluent samples were thawed, mixed, and injected into a Bruker EMX spectrometer equipped with a Bruker Aquax liquid sample cell. ESR spectra were recorded at room temperature. Typical spectrometer parameters were as follows: scan range, 100 G; field set, 3500 G; time constant, 5.12 ms; scan time, 5.12 seconds; modulation amplitude, 1.0 G; modulation frequency 100 kHz; receiver gain, 6.32x10⁵; and microwave power, 10.0 mW. The ESR spectra were simulated using the software developed by Duling¹³ from the laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences (NIEHS, North Carolina).

Quantification of Free-Radical Concentration
Radical concentrations were quantified as follows: 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) was used as a standard. Concentration versus signal intensity standard curves were obtained by double integrating the TEMPO signal at different concentrations (because the ratio of signal to noise was small at our experimental radical concentration, simulated spectra from the weak signals were used to double integrate and improve the integrated result). The same procedure was performed for sample signals. Based on concentration versus signal intensity curves, sample concentrations were obtained.

Videomicroscopy
Vessels were prepared in a fashion similar to that described above for ESR. Diameter was recorded after development of spontaneous myogenic tone. When necessary endothelin-1 (10^-10 to 5x10^-10 mol/L) was added to achieve 30% to 50% constriction. Flow was produced by changing the heights of the reservoirs in equal and opposite directions to generate a pressure gradient.¹⁴ Intraluminal diameter was measured at a pressure gradient of 20 and 100 cm H₂O. To determine the role of specific mitochondrial electron transfer chain (ETC) sites including complex I, III, or IV in FID, site-specific ETC inhibitors, rotenone, myxothiazol, and cyanide, respectively were used. At the end of each experiment, vessels were maximally dilated with papaverine (10^-4 mol/L). The percent dilation was normalized to the maximal diameter in the presence of papaverine. Dilation to papaverine (10^-7 to 10^-4 mol/L) was measured to test the specificity of the inhibitory effect of rotenone. Dilation to bradykinin (10^-10 to 10^-6 mol/L) was recorded in some vessels to assess endothelial function. Endothelial denudation was achieved with air infusion as described previously.¹⁵

All chemicals were obtained from Sigma Chemical Co. BMPO was synthesized as described previously.¹⁶

Fluorescence Detection of Hydrogen Peroxide and Superoxide
Dichlorodihydrofluorescein (DCFH)⁷ and hydroethidine (HE)¹² were used to evaluate the production of hydrogen peroxide (H₂O₂) and superoxide (O₂^·-), respectively, during flow. Four HCRAs from the same atria were exposed to either no flow (pressurized, 0 gradient), or flow at a pressure gradient of 100 cm H₂O, with or without antagonists. One vessel (not pressurized) served as control. DCFH (5 µmol/L) and HE (5 µmol/L) were added in a light-protected chamber for 30 minutes at 37°C either during flow or static conditions. Vessel segments were then washed with fresh PSS solution, and removed for fluorescence microscopy. DCFH and ethidium bromide were excited at 488 nm and 585 nm, respectively. Fresh untreated (control), and experimental tissues are examined in parallel and images recorded using the same computer-specified gain and intensity settings. Generation of free radicals by flow was compared between vessels with and without endothelium. Images were analyzed for intensity of fluorescence within a user-defined region of the arteriolar segment (maximal traceable area of the central portion of the vessel). Artifactual autofluorescent regions were manually eliminated from analysis. Relative average fluorescence intensity was normalized for surface area and compared between control and experimental vessels.

Statistics
All data are expressed as mean±SEM. Percent dilation was calculated as the percent change from preconstricted diameter to the diameter after agonist or flow (maximal diameter was measured after papaverine (10^-4 mol/L). Data from vessels exposed to flow before and after rotenone treatment were compared using a one-way ANOVA with repeated measures for dose and condition (antagonist exposure). Differences between individual means were determined by Newman-Keuls test. The relative fluorescence of arteries exposed to static condition or flow was compared using a two-way ANOVA. All differences were judged to be significant at the level of P<0.05.

	Results

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Sixty-two HCRAs with a mean maximal passive internal diameter 142±10 µm were used. Patient demographics including diagnoses are summarized in the Table.

View this table: [in this window] [in a new window]   Table 1. Patient Demographics (n=62)

Flow-Induced Free-Radical Production in Human Coronary Resistance Vessels
Because previous studies from our laboratory indicated that H₂O₂ plays a role in flow-mediated dilation,⁷ we first examined whether flow actually produced free radicals, using ESR measurements. In the absence of flow, no signal was detected from the PSS in which vessels were incubated. During flow, a prominent ubisemiquinone signal, a radical specifically originated from mitochondria, and BMPO-hydroxyl adduct (BMPO-OH) signal were observed in the effluent 1 minute after thawing of the collected effluent. Fifty minutes later samples were reanalyzed and a prominent BMPO-OH signal was observed (Figure 1A).

View larger version (23K): [in this window] [in a new window]   Figure 1. A, Sample ESR traces. Without flow (top trace), no signal was detected (vessel simply bathed in PSS). In the presence of flow, prominent ubisemiquinone (filled circle) and hydroxyl radical (BMPO-OH, open circle) signals were observed when the measurement was made 1 minute after thawing the samples (middle trace). Fifty minutes later, a greater BMPO-OH radical signal was detected from the same sample. B, Effect of CAT (1000 U/mL and SOD 150 U/mL) on the production of ubisemiquinone and BMPO-OH radicals. CAT or SOD alone reduced BMPO-OH and ubisemiquinone signals. CAT and SOD together abolished all free-radical signals. C, Quantification of free-radical (ubisemiquinone+BMPO-OH radicals) generation in the presence of flow. CAT reduced the amount of free-radical production, which was further decreased by SOD in the presence of CAT.

In separate experiments free-radical scavengers, superoxide dismutase (SOD, 150 U/mL) and/or catalase (CAT, 1000 U/mL), were used. Either CAT or SOD alone reduced both ubisemiquinone and BMPO-OH radical signals (Figure 1B). Total free-radical production (ubisemiquinone+BMPO-OH) during flow is summarized in Figure 1C. The total free-radical production by flow was reduced by either CAT or SOD and was further inhibited by the combination of SOD and CAT. This suggests that BMPO-OH adduct was formed, in part, from the reduction of BMPO-superoxide adduct (BMPO-OH).

The extended duration of flow in these experiments might have led to endothelial damage. Therefore, we examined the dose-response relationship to bradykinin before and after the same shear protocol described (including BMPO). Similar dilations to bradykinin at concentration of 10^-10, 10^-8, and 10^-6 mol/L were observed in vessels before (20±8%, 34±7%, and 71±5%) and after (21±4%, 39±1%, and 73±8%) effluent collection protocol ( n=3, P=NS). Because BK is an endothelium-dependent dilator in these vessels,¹⁷ we conclude that endothelial function is not impaired by the flow protocol used in these experiments.

To determine whether the magnitude of flow-induced dilation is reproducible, flow (20 and 100 cm H₂O) was repeated twice with an intervening 30-minute interval. The magnitude of dilation to both flow stimuli was similar (21±7% versus 20±7% and 70±5% versus 69±5% at 20 and 100 cm H₂O, respectively), indicating that there is no "run-down" with time.

Figure 2A compares O₂^·- and H₂O₂ formation in response to flow before and after endothelial denudation. Flow increased fluorescence intensity in vessels with intact endothelium (+E). The enhanced fluorescence intensity was diminished after endothelial denudation (-E). Average fluorescence ratios, normalized to the control artery, are summarized in Figure 2B. Only flow +E stimulated the production of O₂^·- and H₂O₂ (no flow 1.06±0.1, 0.9±0.1; flow +E 1.38±0.7, 1.42±0.24; flow -E 0.9±0.08, 0.83±0.17), suggesting an important role of endothelium in flow-induced free-radical formation.

View larger version (39K): [in this window] [in a new window]   Figure 2. A, Sample images comparing fluorescence intensity of hydroethidine (HE) and dichlorodihydrofluorescein (DCFH) representing the production of O2·- and H2O2, respectively. Isolated HCRAs exposed to no flow, flow (100 cm H2O pressure gradient) with intact endothelium (+E), or flow without endothelium (-E) were examined. Both HE and DCFH fluorescence intensities were markedly increased in HCRA exposed flow with intact endothelium. Fluorescence intensities were greatly reduced after endothelial denudation in HCRA exposed to the same pressure gradient. Dotted lines indicate edge of vessel wall. B, Summary of fluorescence intensities (normalized to control) in 6 arteries of each group. Flow-induced increases in HE and DCFH fluorescence were observed only in vessels with intact endothelium.

Effect of Complex I Inhibitor of Electron Transport Chain on Flow-Mediated Dilation in Human Coronary Resistance Vessels
To determine whether inhibition of mitochondrial complex I affects flow-mediated dilation in HCRA, dilator responses to flow were compared in the absence and presence of 1 µmol/L rotenone at a pressure gradient of 20 and 100 cm H₂O. Figure 3A shows that rotenone abolished flow-mediated dilation at both pressure gradients (20 cm H₂O: control versus rotenone, 38±5% versus -11±8%; 100 cm H₂O: 74±3% versus 3.2±13%, n=6; P<0.05). However, dilation to papaverine was not affected by rotenone (Figure 3B), indicating that the effect of rotenone is not nonspecific.

View larger version (14K): [in this window] [in a new window]   Figure 3. A, Effect of rotenone on flow-induced dilation. Dilation of HCRAs to increasing pressure gradients was significantly reduced by 1 µmol/L rotenone. B, Dilation to graded doses of papaverine was not altered by rotenone.

Effect of Complex I Inhibitor on Flow-Induced Free-Radical Formation in Human Coronary Resistance Vessels
The effect of the complex I inhibitor, rotenone, on flow-induced free-radical production was also examined by histofluorescence. Flow-induced O₂^·- and H₂O₂ formation was inhibited by 1 µmol/L rotenone (Figure 4A) with average ratios of HE and DCFH fluorescence intensity for arteries before (1.45±0.15 and 1.57±0.12, respectively), and after (0.87±0.17 and 0.95±0.07) treatment with rotenone (Figure 4B; n=6, P<0.05).

View larger version (29K): [in this window] [in a new window]   Figure 4. A, Sample images comparing fluorescence intensity of vessels treated with HE and DCFH, respectively. Isolated HCRAs were exposed to no flow, flow (100 cm H2O pressure gradient), or flow in the presence of 1 µmol/L rotenone. The enhanced HE and DCFH fluorescence intensities in HCRAs by flow were reduced by rotenone. Dotted lines indicate edges of vessel walls. B, Summary of fluorescence intensities (normalized to a freshly isolated nonpressurized control vessel) in 6 arteries for each group. Flow-induced increases in both ethidium bromide and DCFH fluorescence intensities are blocked by rotenone.

The effect of rotenone on O₂^·- and H₂O₂ formation was further quantified by ESR. As indicated in Figure 5, flow-induced free-radical formation was greatly reduced by 1 µmol/L of rotenone (0.27±0.01 µmol/L versus 0.52±0.01 µmol/L, n=6; P<0.05), suggesting an important role of mitochondrial complex I in flow-induced free-radical formation.

View larger version (21K): [in this window] [in a new window]   Figure 5. A, Example of the effect of rotenone on flow-induced ROS production (ESR). Ubisemiquinone and BMPO-OH radical signals produced by flow are greatly attenuated by rotenone. B, Effect of rotenone on flow-induced free-radial production. Rotenone markedly reduced the amount of free radicals generated by flow.

Role of Complex III and IV in Flow-Mediated Dilation
To determine whether inhibition of other respiratory chain sites would affect flow-mediated dilation, diameter changes to flow were compared before and after application of 100 nmol/L myxothiazol,¹⁸ a complex III inhibitor, or 1 µmol/L cyanide,¹⁹ a complex IV inhibitor. Myothiazol partially reduced flow-mediated dilation (20 cm H₂O, 30±2% versus 16±3%; 100 cm H₂O, 67±3% versus 28±4%) (Figure 6A; n=6, P<0.05). Cyanide (1 µmol/L) in a dose shown to reduce metabolic rate,¹⁹ had no effect on flow-mediated dilation (Figure 6B; n=6, P=NS).

View larger version (15K): [in this window] [in a new window]   Figure 6. A, Effect of complex III inhibitor on flow-induced dilation. Dilations of HCRAs to increasing pressure gradients are significantly reduced by 100 nmol/L myxothiazol. B, In contrast, 1 µmol/L cyanide had no effect on flow-induced dilation.

Consistent with our functional studies, similar fluorescence intensities using HE and DCFH were also observed in vessels exposed to flow in the absence and the presence of cyanide. The average ratios of fluorescence intensities (HE and DCFH) relative to the control vessel are as follows: no flow, 1.07±0.04 and 1.17±0.1; flow, 1.63±0.2 and 2.1±0.4; flow+cyanide, 1.62±0.1 and 1.8±0.1 (n=6, each group, P=NS versus flow).

Role of NADPH Oxidase in Flow-Mediated Dilation
The role of NADPH oxidase in flow-induced dilation was also compared before and after application of 3 mmol/L apocynin, an inhibitor of NADPH oxidase (Figure 7). Similar dilator responses to flow were observed in vessels before and after apocynin treatment. The same dose of apocynin has been shown to inhibit O₂^·- production in human internal mammary arteries and saphenous veins.²⁰

View larger version (15K): [in this window] [in a new window]   Figure 7. Effect of apocynin on flow-induced dilation. Dilation of HCRAs to flow is compared before and after incubation with apocynin for 30 minutes. Apocynin (3 mmol/L) had no effect on flow-mediated vasodilation of HCRA.

	Discussion

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The present study demonstrates, for the first time, that mitochondria play an important role in flow-induced dilation in human coronary resistance arteries. The major novel findings are 3-fold. First, flow-induced dilation involves generation of O₂^·- originating from mitochondria. Second, shear stress elicits luminal release of mitochondrial ubisemiquinone, a source for generating O₂^·- and H₂O₂. Third, superoxide generated during shear stress likely arises from metabolic processes occurring between complex I and complex III of the electron transport chain. These findings suggest that in human coronary resistance vessels, flow-induced dilation involves production of H₂O₂, which is formed from O₂^·- generated by mitochondrial respiration. Thus, mitochondrial respiration is necessary for flow-mediated dilation in the human coronary microcirculation.

Shear Stress Elicits Production of Free Radicals
Growing experimental evidence suggests that shear stress stimulates endothelial cell release of free radicals. Hsieh et al²¹ demonstrated that exposure of human umbilical vein endothelial cells to flow for 15 minutes results in an enhanced intracellular ROS, which can be blocked by various antioxidant agents including catalase and 1,3-dimethyl-2-thiourea, scavengers of H₂O₂ and hydroxyl radical, respectively. Increased O₂^·- production was found using a similar preparation by De Keulenaer et al.⁵ We recently showed that H₂O₂ is an EDHF mediating flow-induced dilation in HCRA.⁷ In the present study, production of both O₂^·- and H₂O₂ is increased consistent with this finding.

Mitochondria as a Source of Free Radicals During Flow
The present study identifies the mitochondria as a source of free radicals in response to shear stress. This conclusion is supported by several lines of evidence. First, using ESR, a prominent ubisemiquinone signal, specifically derived from mitochondria,^22,23 was detected in the effluent of perfused vessels. Rotenone, a complex I inhibitor, diminished the ubisemiquinone signal. This strongly suggests that the trapped radical species originated from the electron transport chain of the mitochondria. Ubisemiquinone can serve as an electron donor to O₂ to produce O₂^·-.^24,25 We also observed a prominent BMPO-OH signal that was greatly reduced by catalase, indicating involvement of H₂O₂. Increased HE and DCFH fluorescence intensities representing O₂^·- and H₂O₂ generation were observed in vessels exposed to flow. Second, the enhanced redox fluorescence signal was diminished by rotenone. Finally, FID was either abolished or reduced by the mitochondrial ETC inhibitors, rotenone and myxothiazol. These consistent biochemical, fluorescence, and functional results strongly suggest that mitochondrial-derived ROS mediate FID in HCRA.

Role of Mitochondrial Electron Transport Chain Sites in Free-Radical Production
Electron flow through the mitochondrial ETC occurs by four inner membrane–associated enzyme complexes, with cytochrome c and the mobile carrier ubiquinone.^26–29 NADH derived from both cytosol and mitochondria donates electrons to NADH:ubiquinone oxidoreductase (complex I). Complex I ultimately transfers its electrons to succinate:ubiquinone oxidoreductase (complex II). Electrons from reduced ubiquinone are then transferred to ubiquinol:cytochrome c oxidoreductase (complex III) by the ubisemiquinone radical–generating Q cycle.²³ Electron transport proceeds through cytochrome c, cytochrome c oxidase (complex IV), and finally, molecular oxygen.

Ubisemiquinone serving as an electron donor plays an essential role in generating O₂^·-. Ubisemiquinone (Q^·-) is derived from the reduction of quinone (Q). It reacts with molecular oxygen to form O₂^·- with regeneration of quinone [reaction (1)]:

Ubisemiquinone can also be generated by the reaction of O₂^·- with hydroquinone (HQ) a reduction product of quinone [reaction (2)]:

There are two major mitochondrial ETC regions where ROS are produced: namely, complex I^26,28,29 and complex III.³⁰ To elucidate the location in the ETC responsible for O₂^·- formation during flow, specific inhibitors were examined. Rotenone, a complex I inhibitor, greatly reduced FID but did not affect dilation to papaverine. Consistent with this functional study, rotenone also decreased the ESR ubisemiquinone signal and HE and DCFH fluorescence intensities, suggesting an important role for complex I of the mitochondrial ETC in flow-induced O₂^·- and H₂O₂ generation. Myxothiazol, which inhibits oxidation of ubiquinol to ubisemiquinone in complex III, also reduced FID. In contrast, cyanide, a complex IV inhibitor, had no effect. These findings provide strong evidence that complex I and III of mitochondrial ETC are the major sources of O₂^·- and H₂O₂ that mediate dilation to flow in HCRA. They also support the importance of ubisemiquinone in this process.

In the present study, we observed that ubisemiquinone ESR signals were reduced by SOD. This was not expected and the precise mechanism is not clear. One possible explanation is that by quenching O₂^·-, SOD may disrupt the equilibrium between ubisemiquinone and O₂^·- and shift the reaction toward to the right, thereby decreasing ubisemiquinone. This phenomenon has also been observed by others.³¹ Another possibility is that removal of O₂^·- by SOD decreased ubisemiquinone production by preventing the reaction of hydroquinone (HQ) with O₂^·-.

Alternative Sources for Producing Free Radicals
NADPH oxidase, another putative source for generating O₂^·-, is a membrane-bound flavocytochrome present in many types of cells, including vascular endothelial cells. It uses both flavin and heme groups to shuttle electrons from NADPH to oxygen, yielding O₂^·-. It has been suggested that in cultured human umbilical vein endothelial cell shear stress enhances the activity of the O₂^·--producing NADPH oxidase, which can be inhibited by Diphenylene iodonium (DPI) and Tiron.⁵ DPI nonspecifically inhibits flavoprotein-containing enzymes, including NADPH oxidases, NOS, and complex I of the mitochondrial ETC.³² To examine the involvement of NADPH oxidase in FID, we used apocynin, an inhibitor selective for the NADPH oxidase that acts by inhibiting incorporation of the p47^phox subunit of NADPH oxidase into the membrane unit, thereby inhibiting enzyme function.^20,33 These results indicate a lack of involvement of NADPH oxidase in FID and O₂^·- generation in the human coronary microcirculation.

Study Limitations
There are multiple sources for production of O₂^·- and H₂O₂ in endothelial cells. NOS, cyclooxygenase, and cytochrome P450 are known to be activated by shear stress and may contribute to flow-induced dilation by the elaboration of ROS. Our prior studies indicate that neither NOS or cyclooxygenase products are involved in FMD of coronary arterioles from humans with coronary artery disease.¹⁴ In the present study, rotenone virtually abolished FID, suggesting a significant contribution of mitochondrial free-radical generation to FID in human coronary arterioles.

In addition to augmented production, enhanced oxidative stress may occur by reduced synthesis, inhibition of, or inactivation of endogenous antioxidant systems. Glutathione peroxidase (GPX), SOD, and CAT are among the more important cellular antioxidant defenses. Both GPX and multiple isoforms of SOD can be upregulated by chronic exposure to shear,^34–36 whereas all three systems are modulated by oxidative stress.^37–41 Therefore, the effect of antioxidant activity must be considered in assessing cellular redox states. The short stimulus duration (shear or pharmacology) used in this study would not likely alter antioxidant expression in this tissue.³

Because most microvessels used in this study are derived from patients with CAD, our results may have been skewed toward a prominent role of mitochondrial free radicals acting as an EDHF to compensate for loss of NO in FID. Indeed our laboratory has demonstrated that NO contributes to FMD in subjects without CAD or its risk factors.¹⁴ However, even in these subjects, there is a large NO-independent component of the dilation, possibly due to EDHF. We are not able to examine the role of mitochondria in FID in healthy human coronary resistance arteries. It is possible that in healthy subjects other dilator mechanisms also contribute to FID. This idea is supported by our previous observation of a greater dilation to H₂O₂ in patients with CAD compared with those without.⁷

Coronary vessels used in this study were all derived from human atria. With one exception (response to acetylcholine⁴²), we have observed no differences between vessels from atria and ventricles with respect to dilator and constrictor responses. Most important, flow-induced dilation is similar in both atria and ventricles.¹⁴ Although we cannot be certain, we anticipate that our results can be extrapolated to human ventricular resistance vessels.

It is not known how shear stress signals mitochondrial release of ROS. One potential mechanism may involve changes in the cytoskeletal microfilaments that physically connect sarcolemmal and mitochondrial membranes. The cytoskeleton is key in detecting cellular mechanical deformation.⁴³ Inhibition of polymerization of cytoskeletal actin using cytochalasin D has been shown to reduce pressure-induced myogenic constriction in rat cerebral arteries.⁴⁴ Nocodazole, a blocker of microtubule polymerization, decreases flow-mediated dilation in rat gracilis arteries.⁴⁵

It is also known that mitochondrial function is strongly influenced by attached cytoskeletal filaments and their associated proteins.^46,47 For instance, opening of mitochondrial K_ATP channels and ischemic preconditioning depends on polymerization of actin cytoskeleton.⁴⁸ These observations suggest one mechanism by which shear stress might mechanically link to mitochondrial membrane channels via cytoskeletal filaments to release ROS.

Ubisemiquinone is not a BMPO trapped species. In a preliminary study, we compared the ubisemiquinone signal by ESR in the absence and the presence of BMPO. To our surprise, we were not able to detect a ubisemiquinone signal in the absence of BMPO. No signal was detected in baseline control studies using BMPO alone, excluding the possibility that a nonspecific signal was generated by BMPO. The reason for requiring the presence of BMPO in capturing the ubisemiquinone signal is not clear. One possible explanation is that in biological systems ubisemiquinone may react with other radicals that can be trapped by BMPO, resulting in a radical species (ubisemiquinone+ROS) that is not detected by ESR. In the presence of BMPO, the BMPO trapped radicals no longer react with ubisemiquinone, which may enhance the ubisemiquinone signal into the detectable range.

Clinical Implications
Mitochondria serve as an important source for ROS in many pathological conditions. The vast majority of ROS produced by cells originates from mitochondrial metabolism. The significant contribution of free-radicals to FID in human coronary resistance vessels suggests a mechanism for preserving myocardial perfusion in disease states such as ischemia/reperfusion, diabetes, CAD, and hypertension where ROS generation is increased and dilator mechanisms involving NO may be inhibited.

Summary
Flow increases production of ubisemiquinone and BMPO-OH radicals in human coronary resistance arteries, which can be blocked by rotenone and catalase, respectively. Complex I and III of mitochondrial ETC, but not complex IV, are the major sites for generating free radicals responsible for FID in human coronary arterioles. These results suggest that FID and H₂O₂ formation are the result of O₂^·- generation from mitochondrial respiration.

	Acknowledgments

 
This work was supported by NIH RO1 HL-59238 and P01 HL68769 to D.G., a Veterans Administration Merit Award to D.G., and a Scientist Development Grant from the American Heart Association and NIH RO1 HL067968 to Y.L.

	Footnotes

 
Original received May 15, 2003; revision received August 5, 2003; accepted August 5, 2003.

	References

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