Molecular Mechanisms of Angiotensin II–Mediated Mitochondrial Dysfunction
Linking Mitochondrial Oxidative Damage and Vascular Endothelial Dysfunction
Mitochondrial dysfunction is a prominent feature of most cardiovascular diseases. Angiotensin (Ang) II is an important stimulus for atherogenesis and hypertension; however, its effects on mitochondrial function remain unknown. We hypothesized that Ang II could induce mitochondrial oxidative damage that in turn might decrease endothelial nitric oxide (NO·) bioavailability and promote vascular oxidative stress. The effect of Ang II on mitochondrial ROS, mitochondrial respiration, membrane potential, glutathione, and endothelial NO· was studied in isolated mitochondria and intact bovine aortic endothelial cells using electron spin resonance, dihydroethidium high-performance liquid chromatography –based assay, Amplex Red and cationic dye fluorescence. Ang II significantly increased mitochondrial H2O2 production. This increase was blocked by preincubation of intact cells with apocynin (NADPH oxidase inhibitor), uric acid (scavenger of peroxynitrite), chelerythrine (protein kinase C inhibitor), NG-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor), 5-hydroxydecanoate (mitochondrial ATP-sensitive potassium channels inhibitor), or glibenclamide. Depletion of p22phox subunit of NADPH oxidase with small interfering RNA also inhibited Ang II–mediated mitochondrial ROS production. Ang II depleted mitochondrial glutathione, increased state 4 and decreased state 3 respirations, and diminished mitochondrial respiratory control ratio. These responses were attenuated by apocynin, 5-hydroxydecanoate, and glibenclamide. In addition, 5-hydroxydecanoate prevented the Ang II–induced decrease in endothelial NO· and mitochondrial membrane potential. Therefore, Ang II induces mitochondrial dysfunction via a protein kinase C–dependent pathway by activating the endothelial cell NADPH oxidase and formation of peroxynitrite. Furthermore, mitochondrial dysfunction in response to Ang II modulates endothelial NO· and generation, which in turn has ramifications for development of endothelial dysfunction.
Angiotensin (Ang) II mediates endothelial dysfunction and promotes vascular inflammation and atherogenesis.1–3 Reactive oxygen species (ROS) play key roles in these processes.4,5 In addition to vascular NADPH oxidase–derived ROS, Ang II has been shown to stimulate mitochondrial dysfunction in cardiac, renal, and vascular smooth muscle cells.6–9 Dysfunctional mitochondria, in turn, produce excessive amounts of ROS such as superoxide (), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−).10,11 This overproduction of mitochondrial ROS has been implicated in neurodegenerative diseases, ischemia reperfusion injury, atherosclerosis and aging.12–16 Moreover, recent studies have demonstrated that mitochondrial ROS levels can be decreased by overexpression of either manganese superoxide dismutase or the peroxiredoxins 3 or 5 and that this protects against mitochondrial oxidative damage and dysfunction.17–19 Interestingly, Ang-converting enzyme inhibitors and type I Ang-receptor blockers also reduce age-related mitochondrial dysfunction, attenuate hypertension-induced renal mitochondrial dysfunction, and protect against cardiac mitochondrial dysfunction in the setting of acute ischemia.6–8 These findings suggest that Ang II can alter mitochondrial function; however, the molecular mechanisms underlying this process remain undefined. It also remains to be established whether this effect of Ang II on mitochondrial function occurs in endothelial cells and if this could cause endothelial dysfunction.
We therefore performed this study to examine the effects of Ang II on endothelial cell mitochondria. We used electron spin resonance (ESR) for detection of , H2O2, nitric oxide (NO·), and glutathione (GSH) analysis. Our findings have defined a molecular mechanism for Ang II–mediated mitochondrial dysfunction and demonstrate that this can contribute to the development of vascular endothelial dysfunction.
Materials and Methods
Reagents and Buffers
Apocynin (Apo) and horseradish peroxidase (HRP) were obtained from Calbiochem (San Diego, Calif). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) and Amplex Red were obtained from molecular probes (Carlsbad, Calif). 1-Hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PPH) and bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl)disulfide biradical (RSSR) were purchased from Alexis Corp (San Diego, Calif). Lithium phthalocyanine was a gift from Dr Periannan Kuppusamy (Center for Biomedical EPR Spectroscopy and Imaging, Ohio State University, Columbus). All other reagents were purchased from Sigma-Aldrich (St Louis, Mo).
Measurements of mitochondrial ROS production, membrane potential, and respiratory activity were performed in media A buffer containing 125 mmol/L KCl, 10 mmol/L MOPS, 2 mmol/L MgSO4, 2 mmol/L KH2PO4, 10 mmol/L NaCl, 1 mmol/L EGTA, and 0.7 mmol/L CaCl2, pH 7.2. This buffer was treated with 50 μmol/L desferoxamine to chelate-free iron. Determination of endothelial and NO· production were performed in Krebs–Hepes buffer containing 5.786 g/L NaCl, 0.35 g/L KCl, 0.368 g/L CaCl2, 0.296 g/L MgSO4, 2.1 g/L NaHCO3, 0.142 g/L K2HPO4, 5.206 g/L Na-Hepes, and 2 g/L d-glucose, pH 7.35.
Stock solutions of PPH (10 mmol/L), dissolved in 0.9% NaCl treated with 50 g/L chelex 100 and containing 50 μmol/L desferoxamine and purged with argon, were prepared daily and kept under argon on ice. Desferoxamine was used to decrease autooxidation of PPH catalyzed by trace amounts of free iron. PPH was used in a final concentration of 1 mmol/L.
Cell Culture and Mitochondrial Isolation
Bovine aortic endothelial cells (passage 4 to 8) were cultured on 100-mm plates in medium 199 containing 10% FCS supplemented with 2 mmol/L l-glutamine and 1% vitamins. On the day before the study, the FCS concentration was reduced to 1%. Mitochondria were isolated from confluent BAECs as described previously.20 For details, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Measurement of Mitochondrial ROS Production Using ESR and PPH
Mitochondrial H2O2 was measured using the hydroxylamine PPH in the presence of 5 U/mL HRP and 1 mmol/L acetamidophenol (For details, see the online data supplement).
Measurement of Mitochondrial ROS Production Using Amplex Red
Mitochondrial H2O2 was measured using the HRP-linked Amplex Red fluorescence assay as described previously.21 For details, see the online data supplement.
Determination of Mitochondrial Membrane Potential
The mitochondrial inner membrane electrochemical potential was measured using fluorescence microscopy with JC-1. JC-1 enters the mitochondria in proportion to the membrane potential and forms J-aggregates at the higher intramitochondrial concentrations induced by higher mitochondrial membrane potential (ΔΨ) values.9 For details, see the online data supplement.
Assessment of Mitochondrial Respiratory Activity
Endothelial cell mitochondrial respiratory activity was measured using ESR spectroscopy–based oximetry with lithium phthalocyanine microcrystals.22 For details, see the online data supplement.
Quantification of Mitochondrial Low-Molecular-Weight Thiols
Mitochondrial low-molecular-weight reduced thiols, which are largely composed of GSH, were measured by an ESR-based method using a biradical spin label carrying a disulfide bond, ·RS-SR·.23 For details, see the online data supplement.
Measurements of Nitric Oxide and Intracellular
Nitric oxide production by BAECs was measured using Fe(DETC)2 as described previously.24,25 Intracellular generation was quantified using dihydroethidium and high-performance liquid chromatography–based assay as described previously.26
Small Interfering RNA Construction and Transfection
A detailed description of the sip22phox construction and transfection assay is available in the online data supplement.
Immunoblot Analysis of GADPH, COX, and p22phox
A detailed description of the cellular fractionation and immunoblotting assay is available in the online data supplement.
All data are expressed as means±SEM. Comparisons between groups of treatments were made by 1-way ANOVA, followed by Bonferroni post hoc test when significance was indicated. Values of P<0.05 were considered significant.
Mitochondrial H2O2 Production in Ang II–Treated BAECs
Ang II (200 nmol/L, 4 hours) treatment caused a 2-fold increase in mitochondrial H2O2 production in the presence of malate plus glutamate and a 1.5-fold increase in H2O2 in the presence of succinate, as determined by ESR (Figure 1A and 1B). This increase was inhibited by preincubation of endothelial cells with Apo and chelerythrine, indicating that the Ang II effect on mitochondrial ROS was likely mediated through NADPH oxidase and protein kinase C (PKC). We further confirmed this observation using the Amplex Red assay. Consistent with the ESR data, this assay also detected a significant increase in mitochondrial H2O2 production, and this was completely prevented by preincubation with Apo (Figure 1C and 1D). For subsequent experiments, we used our modified ESR technique for H2O2 measurements because it readily quantifies H2O2 and works in light-disperse samples, where fluorescence detection is limited.
To prove the association between Ang II–induced increase in NADPH oxidase activity and mitochondrial ROS production, we reduced endothelial levels of p22phox, an integral subunit of the NADPH oxidase system using small interfering (si)RNA. Endothelial cells were treated with sip22phox or a nonsilencing control (scrambled) siRNA for 3 days and then stimulated with Ang II for 4 hours. Depletion of p22phox completely blocked Ang II–mediated increase in mitochondrial H2O2. In contrast, treatment with scrambled siRNA did not attenuate the effect of Ang II on mitochondrial H2O2 production (Figure 2A and 2B). Interestingly, depletion of p22phox decreased production of mitochondrial H2O2 in nonstimulated cells, suggesting that the NADPH oxidase modulates baseline production of mitochondrial H2O2 in vascular endothelial cells.
Recent research suggests that peroxynitrite damages mitochondrial respiratory complexes, leading to increased mitochondrial ROS production.27,28 To determine the role of peroxynitrite in Ang II–mediated mitochondrial dysfunction, cells were treated with either NG-nitro-l-arginine methyl ester (L-NAME) to prevent nitric oxide synthesis or uric acid to scavenge peroxynitrite. Both L-NAME and uric acid blocked the increase in mitochondrial H2O2 induced by Ang II. These data implicate peroxynitrite as a mediator of Ang II–induced mitochondrial dysfunction (Figure 1A and 1B).
In vascular smooth muscle cells, mitochondrial ATP-sensitive potassium channels (mitoKATP) have been implicated in Ang II–induced mitochondrial ROS production.9 To determine whether mitoKATP opening contributes to Ang II–mediated mitochondrial ROS production in endothelial cells, intact cells were exposed to 5-hydroxydecanoate (5-HD) (100 μmol/L) or glibenclamide (20 μmol/L), specific and nonspecific inhibitors of mitoKATP, respectively. 5-HD or glibenclamide completely prevented the increase in mitochondrial H2O2 production in Ang II–treated cells while having no effect in mitochondria isolated from control cells (Figure 1A and 1B). Interestingly, applying 5-HD or glibenclamide before Ang II stimulation or during the last 30 minutes of incubation with Ang II had similar protective effects, suggesting that these mitoKATP inhibitors not only prevent Ang II–induced mitochondrial dysfunction but also reverse it.
The role of K+ exchange across the intermembrane space in Ang II–mediated mitochondrial ROS production is further substantiated using the K+/H+ antiporter nigericin. In control mitochondria fueled with malate and glutamate or succinate, nigericin stimulates K+ influx into the matrix and significantly increased ROS production (Figure 2C and 1⇑D). However, in mitochondria isolated from Ang II–treated endothelial cells and fueled with succinate, nigericin significantly decreased ROS production (Figure 2D). These findings strongly suggest that K+ influx/efflux across the inner membrane plays an essential role in regulating mitochondrial ROS production in vascular endothelial cells.
The role of mitochondrial permeability transition pores opening in Ang II–mediated ROS production was also investigated using cyclosporine A. Supplementation of mitochondria isolated from Ang II–treated endothelial cells with cyclosporine A significantly but not completely inhibited Ang II–induced mitochondrial ROS generation (Figure 2C and 2D). Meanwhile, cyclosporine A did not affect H2O2 production in control mitochondria. These data are in line with the previous report by Eliseev et al and suggest that matrix swelling attributable to mitochondrial permeability transition opening partially contributes to ROS production in response to Ang II.29
It is important to note that the NADPH oxidase subunit p22phox was detected exclusively in the membrane fraction but not in isolated mitochondria (Figure 1F). This substantiates the purity of our mitochondrial preparations and verifies that the source of H2O2 measured in the presence of complex I and II substrates is the respiratory chain of mitochondria rather than the NADPH oxidase. Furthermore, Western blots indicated that our mitochondrial fraction did not contain the cytoplasmic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 1D).
Addition of Ang II or the PKC activator 4-phorbol 12-myristate 13-acetate directly to isolated mitochondria did not have any effect on mitochondrial H2O2 (Figure 2E and 2F).
Effect of Ang II on ΔΨ in Endothelial Cells
ΔΨ is essential for normal mitochondrial function. We therefore examined the effect of Ang II on this mitochondrial parameter. Treatment of BAECs with Ang II (200 nmol/L, 4 hours) depolarized ΔΨ (Figure 3A and 3B). These changes were blunted by pretreatment with Apo, uric acid, ebselen, 5-HD, or glibenclamide. As described previously, the mitoKATP opener diazoxide caused a substantial drop in the mitochondrial membrane potential. These findings indicate that depolarization of ΔΨ by Ang II was dependent on NADPH oxidase activity and ROS and involved opening of mitoKATP channels.
Effect of Ang II on Mitochondrial Respiration
Treatment of BAECs with Ang II (200 nmol/L) for 4 hours significantly increased mitochondrial O2 consumption during state 4 respiration (in the absence of ADP). In contrast, Ang II decreased state 3 respiration (in the presence of ADP). The respiratory control ratio (RCR), calculated as the state 3/state 4 ratio, under both substrate conditions was therefore markedly reduced by Ang II, indicating uncoupling between mitochondrial respiration and oxidative phosphorylation (Table).
As noted above, the increase in mitochondrial H2O2 production caused by Ang II was reduced by Apo. To determine whether the effect of Ang II on mitochondrial respiration was also caused by NADPH oxidase activation, we pretreated BAECs with 600 μmol/L Apo for 30 minutes before administration of Ang II. This attenuated the Ang II–induced increase in state 4 respiration with malate plus glutamate as substrate and abolished the Ang II–induced decrease in state 3 respiration with succinate as substrate. Furthermore, treatment of cells with mitoKATP inhibitors 5-HD or glibenclamide significantly improved respiration (Table). These data demonstrate that Ang II decreases mitochondrial RCR by activating NADPH oxidase and mitoKATP.
Effect of Ang II on Mitochondrial Reduced GSH
A consequence of oxidative stress is depletion of GSH and other small molecule thiols. Incubation with 200 nmol/L Ang II for 4 hours significantly decreased the intramitochondrial concentrations of low-molecular-weight thiols, and this was prevented by pretreatment with Apo, 5-HD, or glibenclamide (Figure 4A). Concurrently, Ang II decreased cytosolic GSH (Figure 4B). These findings indicate that Ang II depletes not only cytosolic but also intramitochondrial GSH content, which further enhances Ang II–mediated mitochondrial oxidative damage.
Effects of Ang II–Mediated Mitochondrial Dysfunction on Endothelial Superoxide Production
To further examine the effects of mitochondrial dysfunction on endothelial oxidative stress, intracellular production was examined by DHE and a high-performance liquid chromatography–based assay in intact endothelial cells. In Ang II–treated cells, there was a 1.3-fold increase in production over the control measurements. Preincubation of BAECs with uric acid, Apo and chelerythrine abolished Ang II–induced in production (Figure 5). The mitoKATP channels inhibitor 5-HD added during the last 30 minutes of the 4-hour incubation with Ang II brought back endothelial production to baseline level. The decline in production with 5-HD implies that mitochondrial dysfunction enhances endothelial oxidative stress.
Effect of Ang II–Mediated Mitochondrial Dysfunction on Endothelial NO Bioavailability
Because of the importance of endothelial NO· in vascular function and the rapid inactivation of NO· by , we tested the hypothesis that 5-HD could prevent Ang II–induced decrease in NO· bioavailability in endothelial cells. Production of NO· in BAECs was determined using the NO·-specific colloid spin trap Fe-(DETC)2.24 Ang II treatment for 4 hours significantly decreased endothelial cell NO· production compared with control cells and this was prevented by either 5-HD or uric acid (Figure 6). Thus, blocking mitoKATP with 5-HD completely prevented the decline in endothelial NO· caused by Ang II, suggesting that mitochondria plays a major role in Ang II–induced endothelial dysfunction.
The present study provides the first evidence that Ang II mediates mitochondrial dysfunction in vascular endothelial cells. Ang II increased mitochondrial ROS production and decreased mitochondrial ΔΨ, RCR, and low molecular weight thiols content. These deleterious effects of Ang II on mitochondrial function were associated with increased cellular production and decreased endothelial NO· bioavailability. Furthermore, our results indicate that Ang II–mediated mitochondrial dysfunction is dependent on activation of vascular NADPH oxidases and opening of the mitoKATP channels. Inhibition of NADPH oxidases and PKC by Apo and chelerythrine respectively, completely prevented mitochondrial dysfunction induced by Ang II. Treatment with the mitoKATP channels blocker 5-HD and glibenclamide prevented the increase in mitochondrial H2O2 production and the decrease in mitochondrial ΔΨ, RCR, and low molecular weight thiols content induced by Ang II. Uric acid and L-NAME prevented the effects of Ang II on mitochondrial function, suggesting that ONOO− has an important role in mediating this dysfunction.
Mitochondrial can be produced by complexes I, II and III of the electron transport chain and released primarily toward the mitochondrial matrix.21,30 Given the abundance of manganese superoxide dismutase in the matrix, is rapidly dismutated to H2O2, which in turn readily diffuses out of the mitochondria and was measured by the acetamidophenol-HRP-PPH based ESR assay.31 Our studies indicate that Ang II significantly increases the production of mitochondrial H2O2 in vascular endothelial cells. Interestingly, the Ang II–mediated increase in mitochondrial H2O2 was associated with a decrease in mitochondrial ΔΨ. The higher rate of ROS production in depolarized mitochondria suggests that Ang II effect does not depend on reverse electron flow from complex II to complex I. Rather; this phenomenon could potentially be explained by direct oxidative damage to complexes I and II. Oxidative damage to these complexes has been previously shown to increase mitochondrial ROS production.21
NADPH oxidase activation is an early response of endothelial cells to Ang II.32 Ang II binds to the Ang II type 1 receptor, leading to rapid-generation of ROS through PKC-dependent activation of NADPH oxidase. Apocynin is known to block the activation of NADPH oxidase, and chelerythrine selectively inhibits PKC. Both inhibitors dramatically attenuated mitochondrial ROS generation in response to Ang II. We also demonstrated that Apo attenuated the Ang II–induced decrease in mitochondrial RCR, ΔΨ and low molecular weight thiols. Most importantly, depletion of p22phox with siRNA led to a significant decrease in ROS production in mitochondria isolated from Ang II treated cells. Taken together, these results suggest that mitochondrial dysfunction by Ang II requires the full enzymatic activity of NADPH oxidase.
Our data also clearly implicate mitoKATP channels in Ang II–mediated mitochondrial dysfunction. As we observed, 5-HD, a specific inhibitor of mitoKATP channels, and glibenclamide, a nonselective inhibitor of mitoKATP channels suppressed Ang II–induced mitochondrial H2O2 production, ΔΨ depolarization and prevented the decrease in mitochondrial RCR and reduced thiol content. The mechanism by which Ang II regulates mitoKATP channels activity is unclear. An involvement of and PKC in activation of mitoKATP channels has been suggested in vascular smooth muscle cells and cardiac cells.9,33 Ang II activated NADPH oxidase–derived is capable of stimulating the opening of the mitoKATP channels via a direct action on the sulfhydryl groups of this channel.34 Opening of these channels has been proposed to increase K+ influx causing matrix alkalinization, swelling, mild uncoupling, and ROS production.35 The critical role of K+ influx across the inner membrane is further demonstrated in our experiments with the K+/H+ antiporter nigericin.
Of interest, the K+ flux catalyzed by mitoKATP opening is so small that it is estimated to cause a decrease in mitochondrial ΔΨ of only 1 to 2 mV.35 For this reason, we believe that the significant Ang II–mediated decrease in ΔΨ is not solely attributable to the K+ influx across these channels. Taken into consideration, the partial inhibition of mitochondrial ROS production with cyclosporine A and findings from other laboratories, it is reasonable to speculate that matrix swelling associated with opening of these channels and superoxide-mediated activation of uncoupling proteins through lipid peroxidation products and reactive aldehydes are the most probable mechanisms to induce proton leak across the inner membrane, suppressing mitochondrial ΔΨ.35–37 These questions need to be clarified by further studies.
rapidly reacts with NO· forming ONOO−, which, in turn, can cause mitochondrial damage by several mechanisms that include membrane lipid peroxidation and damage to respiratory complexes I, III, IV, and V.27,28 Vatassery et al and Brookes et al reported that ONOO− treatment of rat brain mitochondria resulted in an increase in state 4 respiration and a decrease in state 3 respiration, leading them to suggest that ONOO− results in leakage of protons across the inner mitochondrial membrane and uncoupling of oxidative phosphorylation.38,39 Recently, ONOO− has been shown to inactivate mitochondrial complexes possibly via tyrosine nitration.40 In keeping with these effects of ONOO−, we found that uric acid and L-NAME both protect against Ang II–mediated mitochondrial dysfunction. Prior evidence obtained from rat brain submitochondrial particles suggests that the damaging effects of ONOO− on the respiratory complexes are totally prevented in the presence of GSH.41 In this context, Ang II significantly reduced mitochondrial concentrations of GSH. It is interesting to speculate that initial oxidative events caused by Ang II resulted in a decrease of GSH, which in turn could sensitize the mitochondria to ONOO− and other oxidants.
The above discussed changes in mitochondrial function had a significant effect on total endothelial cell ROS production, as is evidenced by the fact that Ang II increased endothelial levels and that this was attenuated by 5-HD. Interestingly, Apo blocked the increase in endothelial production caused by Ang II, suggesting a role for the NADPH oxidase. Our studies of isolated mitochondria strongly suggest that ROS produced by the NADPH oxidase can alter mitochondrial function and increase mitochondrial ROS production. It is also possible that H2O2, which diffuses out of the mitochondria, can diffuse to the cytoplasm where it could play a role in activating the NADPH oxidase in a feed-forward fashion. Indeed, Li et al have shown that the NADPH oxidase can be stimulated by H2O2 and lipid peroxides.42
One of the consequences of an increase in endothelial production is a decline of endothelial NO· bioavailability. Accordingly, we found that 4-hour exposure to Ang II decreased levels of NO· and that this was completely prevented by 5-HD. To our knowledge this is the first evidence that the mitochondria are involved in NO· loss in response to Ang II. The present data provide a framework linking mitochondrial damage to endothelial dysfunction and afford additional insight into how Ang II contributes to vascular pathophysiology.
Based on our findings, we suggest the following hypothesis (Figure 7). Ang II stimulates NADPH oxidase via PKC-dependent pathways, which in turn increases ROS production. can then directly activate the mitoKATP channels or react with NO· to form ONOO−, which can damage respiratory complexes, leading to mitochondrial dysfunction. Through a positive-feedback loop, the increased mitochondrial H2O2 production can lead to further activation of cellular NADPH oxidase, resulting in increased intracellular production and diminished NO· bioavailability. The novel actions of Ang II on mitochondrial function raise the possibility that mitochondria-targeted antioxidants might prevent endothelial dysfunction and be beneficial in diseases such as hypertension and atherosclerosis.
We are indebted to Dr Periannan Kuppusamy for providing us with the lithium phthalocyanine oximetry microcrystals.
Sources of Funding
This work was supported by funding from NIH grants P0-1 HL058000 and P0-1 HL075209 and American Heart Association Grant SDG 0430201N.
Original received December 1, 2007; resubmission received August 28, 2007; revised resubmission received December 6, 2007; accepted December 10, 2006.
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