Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.     

Sirtuin3 Dysfunction Is the Key Determinant of Skeletal Muscle Insulin Resistance by Angiotensin II

BackgroundAngiotensin II promotes insulin resistance. The mechanism underlying this abnormality, however, is still poorly defined. In a different setting, skeletal muscle metabolism and insulin signaling are regulated by Sirtuin3.ObjectiveHere, we investigate whether angiotensin II-induced insulin resistance in skeletal muscle is associated with Sirtuin3 dysregulation and whether pharmacological manipulation of Sirtuin3 confers protection.ResultsAngiotensin II promotes insulin resistance in skeletal muscle cells via mitochondrial oxidative stress, resulting in a two-fold increase in superoxide generation. In this context, reactive oxygen species open the mitochondrial permeability transition pore and significantly lower Sirtuin3 levels and activity impairing the cell antioxidant defense. Angiotensin II-induced Sirtuin3 dysfunction leads to the impairment of AMP-activated protein kinase/nicotinamide phosphoribosyltransferase signaling. Acetyl-L-carnitine, by lowering angiotensin II-induced mitochondrial superoxide formation, prevents Sirtuin3 dysfunction. This phenomenon implies the restoration of manganese superoxide dismutase antioxidant activity and AMP-activated protein kinase activation. Acetyl-L-carnitine protection is abrogated by specific Sirtuin3 siRNA.ConclusionsOur data demonstrate that angiotensin II-induced insulin resistance fosters mitochondrial superoxide generation, in turn leading to Sirtuin3 dysfunction. The present results also highlight Sirtuin3 as a therapeutic target for the insulin-sensitizing effects of acetyl-L-carnitine.     

All-trans retinoic acid prevents angiotensin II- and mechanical stretch-induced reactive oxygen species generation and cardiomyocyte apoptosis

Cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodeling to heart failure. All-trans retinoic acid (RA), a bioactive vitamin A derivative, prevents stretch- and angiotensin II (Ang II)-induced cardiac hypertrophy. However, the anti-apoptotic potential of RA in the heart remains unexplored. Here, we demonstrate that stretch- and Ang II-induced apoptosis is prevented by RA in neonatal cardiomyocytes. RA improved mitochondrial function by inhibiting the stretch- and Ang II-induced reduction in mitochondrial membrane potential, cytochrome c release and by increasing the Bcl2/Bax ratio. RA inhibited stretch- and Ang II-induced intracellular reactive oxygen species (ROS) generation and upregulated the SOD2 level. Hydrogen peroxide-induced increases in the number of TUNEL-positive cells and percentage of Annexin V positive cells, were dose-dependently inhibited by RA. The thiol antioxidant, N-acetyl cysteine (NAC), completely inhibited stretch- and Ang II-induced apoptosis. Using diazoxide (mitochondrial ATP-sensitive K(+) channel opener) and SDS (NADPH oxidase activator), we confirmed that RA suppressed both mitochondrial- and NADPH oxidase-derived ROS. We also observed that both RAR and RXR were involved in preventing Ang II- and stretch-induced ROS production and apoptosis, by using selective retinoid receptor agonists and antagonists. Our data provide the first evidence that RA prevents Ang II and stretch induced apoptosis, by inhibiting ROS generation and increasing the anti-oxidant defense system, suggesting that RA-mediated signaling may provide a new therapeutic target for the prevention of the cardiac remodeling process.     

Angiotensin II-induced mitochondrial Nox4 is a major endogenous source of oxidative stress in kidney tubular cells

Angiotensin II (Ang II)-induced activation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase leads to increased production of reactive oxygen species (ROS), an important intracellular second messenger in renal disease. Recent findings suggest that Ang II induces mitochondrial depolarization and further amplifies mitochondrial generation of ROS. We examined the hypothesis that ROS injury mediated by Ang II-induced mitochondrial Nox4 plays a pivotal role in mitochondrial dysfunction in tubular cells and is related to cell survival. In addition, we assessed whether angiotensin (1-7) peptide (Ang-(1-7)) was able to counteract Ang II-induced ROS-mediated cellular injury. Cultured NRK-52E cells were stimulated with 10(-6) M Ang II for 24 h with or without Ang-(1-7) or apocynin. Ang II simulated mitochondrial Nox4 and resulted in the abrupt production of mitochondrial superoxide (O(2) (-)) and hydrogen peroxide (H(2)O(2)). Ang II also induced depolarization of the mitochondrial membrane potential, and cytosolic secretion of cytochrome C and apoptosis-inducing factor (AIF). Ang-(1-7) attenuated Ang II-induced mitochondrial Nox4 expression and apoptosis, and its effect was comparable to that of the NAD(P)H oxidase inhibitor. These findings suggest that Ang II-induced activation of mitochondrial Nox4 is an important endogenous source of ROS, and is related to cell survival. The ACE2-Ang-(1-7)-Mas receptor axis should be investigated further as a novel target of Ang II-mediated ROS injury.     

Chronically elevated glucose compromises myocardial mitochondrial DNA integrity by alteration of mitochondrial topoisomerase function

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) mutations are also associated with different types of cancer and neurodegenerative diseases. The goal of this study was to determine if chronically elevated glucose increase in mtDNA damage contributed to mitochondrial dysfunction and identify the underlying basis for mtDNA damage. H9c2 myotubes (a cardiac-derived cell line) were studied in the presence of 5.5, 16.5, or 33.0 mM glucose for up to 13 days. Tests of mitochondria function (Complex I and IV activity and ATP generation) were all significantly depressed by elevated media glucose. Intramitochondrial superoxide and intracellular superoxide levels were transiently increased during the experimental period. AnnexinV binding (a marker of apoptosis) was significantly increased after 7 and 13 days of high glucose. Thirteen days of elevated glucose significantly increased mtDNA damage globally and across the region encoding for the three subunits of cytochrome oxidase. Using mitochondria isolated from cells chronically exposed to elevated glucose, we observed significant increases in topoisomerase-linked DNA cleavage. Mitochondria-dependent DNA cleavage was significantly exacerbated by H(2)O(2) and that immunoprecipitation of mitochondrial extracts with a mtTOP1 antibody significantly decreased DNA cleavage, indicating that at least part of this activity could be attributed to mtTOP1. We conclude that even mild increases in glucose presentation compromised mitochondrial function as a result of a decline in mtDNA integrity. Separate from a direct impact of oxidative stress on mtDNA, ROS-induced alteration of mitochondrial topoisomerase activity exacerbated and propagated increases in mtDNA damage. These findings are significant in that the activation/inhibition state of the mitochondrial topoisomerases will have important consequences for mitochondrial DNA integrity and the well being of the myocardium.     

Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.     

Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells

Oxidant-induced death and dysfunction of pulmonary vascular cells play important roles in the evolution of acute lung injury. In pulmonary artery endothelial cells (PAECs), oxidant-mediated damage to mitochondrial DNA (mtDNA) seems to be critical in initiating cytotoxicity inasmuch as overexpression of the mitochondrially targeted human DNA repair enzyme, human Ogg1 (hOgg1), prevents both mtDNA damage and cell death (Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, and Gillespie MN. Am J Physiol Lung Cell Mol Physiol 283: L205-L210, 2002). The mechanism by which mtDNA damage leads to PAEC death is unknown, and the present study tested the specific hypothesis that enhanced mtDNA repair suppresses PAEC mitochondrial dysfunction and apoptosis evoked by xanthine oxidase (XO). PAECs transfected either with an adenoviral vector encoding hOgg1 linked to a mitochondrial targeting sequence or with empty vector were challenged with ascending doses of XO plus hypoxanthine. Quantitative Southern blot analyses revealed that, as expected, hOgg1 overexpression suppressed XO-induced mtDNA damage. Mitochondrial overexpression of hOgg1 also suppressed the XO-mediated loss of mitochondrial membrane potential. Importantly, hOgg1 overexpression attenuated XO-induced apoptosis as detected by suppression of caspase-3 activation, by reduced DNA fragmentation, and by a blunted appearance of condensed, fragmented nuclei. These observations suggest that mtDNA damage serves as a trigger for mitochondrial dysfunction and apoptosis in XO-treated PAECs.     

NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

ClC-3 deficiency prevents apoptosis induced by angiotensin II in endothelial progenitor cells via inhibition of NADPH oxidase

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. It is reported that the circulating EPCs number is decreased during hypertension. However, the detailed mechanism is still unclear. Our previous studies have shown that ClC-3 chloride channel is up-regulated with the development of hypertension. This study aims to test whether ClC-3 participates in EPC apoptosis under the condition of increased oxidative stress in angiotensin II (Ang II)-induced hypertension. The results showed that stimulation with 10^?6mol/L Ang II significantly up-regulated the endogenous ClC-3 expression and increased intracellular reactive oxygen species (ROS) generation in EPCs of wild type mice, accompanied by an enhanced NADPH oxidase activity and the expression of gp91^phox (NOX-2), a key catalytic subunit of NADPH oxidase. However, these effects of Ang II were significantly reduced in EPCs of ClC-3^?/? mice. Compared with control, treatment with Ang II induced EPCs apoptosis in wild type mice, concomitantly with declined Bcl-2/Bax ratio, depressed mitochondrial membrane potential and activation of poly(ADP-ribose) polymerase, which was remarkably prevented by both ClC-3 knockout and NADPH oxidase inhibitor apocynin. In addition, the role of ClC-3 deficiency in protecting EPCs against Ang II-induced oxidative stress and apoptosis was further confirmed in Ang II-infused hypertensive mice in vivo. In conclusion, ClC-3 deficiency inhibited Ang II-induced EPC apoptosis via suppressing ROS generation derived from NADPH oxidase.     

Mechanism of apoptosis induced by a new topoisomerase inhibitor through the generation of hydrogen peroxide

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.     

Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.     

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (rho0 cells). However, unlike the case with rho+ cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic rho0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.     

Endothelin mediates superoxide production in angiotensin II-induced hypertension in rats

Angiotensin II and endothelin-1 (ET) are two hormones involved in cardiovascular diseases and well known for their capacity to induce free radical generation in vascular and cardiac tissues. In addition to its prooxidative effect, angiotensin II can increase the synthesis of ET-1 in vascular smooth muscle cells (VSMC). Our objective was to determine whether the ET-1 synthesis in VSMC is involved in angiotensin II-induced superoxide anion production in rats. Our results show that treatments of isolated VSMC with angiotensin II and ET increased superoxide. However, this increase occurred in a bimodal fashion for angiotensin II with a fast transient production (10 min) and a late sustained production (6 h), while ET-1 induced superoxide formation after a delay of 6 h. LU302872 and BQ-123, a nonselective and a selective ETA receptor antagonists, respectively, prevented angiotensin II-induced superoxide anion production only during the late phase. In contrast, BQ-3020, a selective ETB receptor antagonist, had no effect. In vivo, LU302872 reduced the aortic superoxide production induced by angiotensin II administered for 12 days. In conclusion, our results suggest that the superoxide generation induced by chronic angiotensin II infusion may be mediated by ET-1 acting on ETA receptors in VSMC in vitro. Furthermore, this effect appears to contribute to the excess superoxide production during the chronic activation of the renin-angiotensin system in vivo.     

Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II: ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES*

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.     

Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide O2. radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I. Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to an increase in O2. generation and sensitization of the leukemia cells to anticancer agents whose action involve free radical generation. Using leukemia cells with genetic alterations in mitochondrial DNA and biochemical approaches, we further demonstrated that As2O3, a clinically active anti-leukemia agent, inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another O2.-generating agent against cultured leukemia cells and primary leukemia cells isolated from patients. Our study shows that interfering mitochondrial respiration is a novel mechanism by which As2O3 increases generation of free radicals. This novel mechanism of action provides a biochemical basis for developing new drug combination strategies using As2O3 to enhance the activity of anticancer agents by promoting generation of free radicals.     

Mitochondrial superoxide decreases yeast survival in stationary phase

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.     

Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance depend on coordinated expression of genes in the nucleus and mitochondria. A variety of intracellular and extracellular signals transmitted by hormones and second messengers have to be integrated to provide mammalian cells with a suitable abundance of mitochondria and mtDNA to meet their energy demand. It has been proposed that reactive oxygen species (ROS) and free radicals generated from respiratory chain are involved in the signaling from mitochondria to the nucleus. Increased oxidative stress may contribute to alterations in the abundance of mitochondria as well as the copy number and integrity of mtDNA in human cells in pathological conditions and in aging process. Within a certain level, ROS may induce stress responses by altering expression of specific nuclear genes to uphold the energy metabolism to rescue the cell. Once beyond the threshold, ROS may cause oxidative damage to mtDNA and other components of the affected cells and to elicit apoptosis by induction of mitochondrial membrane permeability transition and release of pro-apoptotic proteins such as cytochrome c. On the basis of recent findings gathered from this and other laboratories, we review the alterations in the abundance of mitochondria and mtDNA copy number of mammalian cells in response to oxidative stress and the signaling pathways that are involved.