Reactive oxygen species as mediators of cellular senescence

Aging has often been viewed as a random process arising from the accumulation of both genetic and epigenetic changes. Increasingly, the notion that aging is a stochastic process is being supplanted by the concept that maximum lifespan of an organism is tightly regulated. This knowledge has led to a growing overlap between classical signal transduction paradigms and the biology of aging. We review certain specific examples where these seemingly disparate disciplines intersect. In particular, we review the concept that intracellular reactive oxygen species function as signalling molecules and that oxidants play a central role as mediators of cellular senescence.     

Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion

Signal transduction by reactive oxygen species (ROS; "redox signaling") has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. A variety of mitogenic signals, including those released by receptor tyrosine kinase (RTKs) ligands and oncogenic H-Ras, involve as a critical downstream event the intracellular generation of ROS. Signaling by integrins is also essential for the growth of most cell types and is constantly integrated with growth factor signaling. We provide here evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during fibroblast adhesion and spreading. Moreover, we propose a synergistic action of integrins and RTKs for redox signaling. Integrin-induced ROS are required to oxidize/inhibit the low molecular weight phosphotyrosine phosphatase, thereby preventing the enzyme from dephosphorylating and inactivating FAK. Accordingly, FAK phosphorylation and other downstream events, including MAPK phosphorylation, Src phosphorylation, focal adhesion formation, and cell spreading, are all significantly attenuated by inhibition of redox signaling. Hence, we have outlined a redox circuitry whereby, upon cell adhesion, oxidative inhibition of a protein tyrosine phosphatase promotes the phosphorylation/activation and the downstream signaling of FAK and, as a final event, cell adhesion and spreading onto fibronectin.     

Reactive oxygen species as mediators of angiotensin II signaling

Angiotensin II stimulates a plethora of signaling pathways leading to cell growth and contraction. Recent work has shown that reactive oxygen species are involved in transducing many of the effects of angiotensin II, and are in fact produced in response to agonist-receptor binding. Angiotensin II stimulates a NAD(P)H oxidase to produce superoxide and hydrogen peroxide, both of which may act on intracellular growth-related proteins and enzymes to mediate the final physiological response. Of particular importance is hydrogen peroxide, which mediates angiotensin II stimulation of such important intracellular signals as EGF-receptor transactivation, p38 mitogen activated protein kinase, and Akt. Future work will be directed towards identifying other important redox-sensitive signaling pathways and their relationship to the physiology and pathophysiology of the renin-angiotensin system.     

Reactive oxygen species as mediators of photoreceptor apoptosis in vitro

Retinitis pigmentosa is a heterogeneous group of retinal degenerations characterized by a progressive loss of photoreceptors through the process of apoptosis. The apoptotic cell death of photoreceptors appears to represent a final common pathway in the pathology of retinitis pigmentosa. Previous studies have reported the ability of antioxidants to ameliorate light-induced retinal degeneration, suggesting a role for oxidative stress in photoreceptor cell death. This study demonstrates an early and sustained increase in intracellular reactive oxygen species accompanied by a rapid depletion of intracellular glutathione in an in vitro model of photoreceptor apoptosis. These early changes in the cellular redox state precede disruption of mitochondrial transmembrane potential, nuclear condensation, DNA nicking, and cell shrinkage, all of which are well-characterized events of apoptotic cell death. The ability of zinc chloride and pyrrolidine dithiocarbamate, two established antioxidants, to inhibit photoreceptor apoptosis through the scavenging of intracellular reactive oxygen species establishes a role for reactive oxygen species as possible mediators of in vitro photoreceptor apoptosis. This study provides a molecular basis for the inhibition of photoreceptor apoptosis by antioxidants.     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

Reactive oxygen species as cardiovascular mediators: Lessons from endothelial-specific protein overexpression mouse models

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.     

Reactive oxygen species in cell signaling

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of "oxidative stress" is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.     

Endogenous lectins as mediators of tumor cell adhesion

Endogenous carbohydrate-binding proteins have been found in various normal tissues and cells. Although lectins with different sugar-binding specificities have been described, the most prevalent ones are those that bind beta-galactosides. The ability of some normal and malignant cells to bind exogenous carbohydrate-containing ligands suggested that lectinlike activity is associated with the cell surface and that carbohydrate-binding proteins might mediate intercellular recognition and adhesion. We found that extracts of various cultured murine and human tumor cells exhibit a galactoside-inhibitable hemagglutinating activity. This activity was associated with two proteins of molecular weights of 34,000 and 14,500 daltons, which were purified by affinity chromatography by using immobilized asialofetuin. That these lectins are present on the cell surface was indicated by the binding of monoclonal antilectin antibodies to the surface of various tumor cells and by the immunoprecipitation of 125I-labeled lectins from solubilized cell-surface iodinated cells by polyclonal antilectin antibodies. That these cell surface lectins are functional was demonstrated by the ability of the galactose-terminating asialofetuin to enhance cell aggregation and of asialofetuin glycopeptides to block this homotypic aggregation as well as to suppress cell attachment to substratum, and by the inhibition of both asialofetuin-induced cell aggregation and cell attachment to substratum by the binding of monoclonal antilectin antibodies to the cell surface. These findings implicate cell surface lectins as mediators of cell-cell and cell-substratum adhesion. Some of these cellular interactions might be important determinants of tumor cell growth and metastasis.     

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H2O2 cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as "risk factors" for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases.     

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H₂O₂ cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as “risk factors” for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases. (Mol Cell Biochem 264: 85–97, 2004)     

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut-derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co-culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA-specific CD4(+) T-cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA-specific T-cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.     

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

The present study illustrates elements ofthe signal cascades involved in the activation of taurine effluxpathways in myotubes derived from skeletal muscle cells. Exposingprimary skeletal muscle cells, loaded with ¹⁴C-taurine, to1) hypotonic media, 2) the phospholipaseA₂ (PLA₂) activator melittin, 3)anoxia, or 4) lysophosphatidyl choline (LPC) causes anincrease in ¹⁴C-taurine release and a concomitantproduction of reactive oxygen species (ROS). The antioxidants butulatedhydroxy toluene and vitamin E inhibit the taurine efflux after cellswelling, anoxia, and addition of LPC. The muscle cells possess twoseparate taurine efflux pathways, i.e., a swelling- andmelittin-induced pathway that requires 5-lipoxygenase activity foractivation and a LPC-induced pathway. The two pathways aredistinguished by their opposing sensitivity toward the anion channelblocker DIDS and cholesterol. These data provide evidence forPLA₂ products and ROS as key mediators of the signalcascade leading to taurine efflux in muscle.

     

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.     

Reactive oxygen species, cell signaling, and cell injury

Oxidative stress has traditionally been viewed as a stochastic process of cell damage resulting from aerobic metabolism, and antioxidants have been viewed simply as free radical scavengers. Only recently has it been recognized that reactive oxygen species (ROS) are widely used as second messengers to propagate proinflammatory or growth-stimulatory signals. With this knowledge has come the corollary realization that oxidative stress and chronic inflammation are related, perhaps inseparable phenomena. New pharmacological strategies aimed at supplementing antioxidant defense systems while antagonizing redox-sensitive signal transduction may allow improved clinical management of chronic inflammatory or degenerative conditions, including Alzheimer's disease. Introduction of antioxidant therapies into mainstream medicine is possible and promising, but will require significant advances in basic cell biology, pharmacology, and clinical bioanalysis.     

Reactive oxygen species and hormonal control of cell death

The accumulation of reactive oxygen species (ROS) is involved in regulating cell death. Pathogen- and ozone-induced processes have become important models for the study of cell death regulation by ROS. Hydrogen peroxide and superoxide have emerged as the two key ROS and recent studies have addressed their sources and control of their production. ROS signals interact directly or indirectly with several other signaling pathways, such as nitric oxide, and the stress hormones salicylic acid, jasmonic acid and ethylene. The interaction and balance of these pathways determines whether the cell lives or dies.     

Reactive oxygen species are critical mediators of coronary collateral development in a canine model

Recent evidence suggests that reactive oxygen species (ROS) promote proliferation and migration of vascular smooth muscle (VSMC) and endothelial cells (EC). We tested the hypothesis that ROS serve as crucial messengers during coronary collateral development. Dogs were subjected to brief (2 min), repetitive coronary artery occlusions (1/h, 8/day, 21 day duration) in the absence (occlusion, n = 8) or presence of N-acetylcysteine (NAC) (occlusion + NAC, n = 8). A sham group (n = 8) was instrumented identically but received no occlusions. In separate experiments, ROS generation after a single 2-min coronary artery occlusion was assessed with dihydroethidium fluorescence. Coronary collateral blood flow (expressed as a percentage of normal zone flow) was significantly increased (71 +/- 7%) in occlusion dogs after 21 days but remained unchanged (13 +/- 3%) in sham dogs. Treatment with NAC attenuated increases in collateral blood flow (28 +/- 8%). Brief coronary artery occlusion and reperfusion caused ROS production (256 +/- 33% of baseline values), which was abolished with NAC (104 +/- 12%). Myocardial interstitial fluid produced tube formation and proliferation of VSMC and EC in occlusion but not in NAC-treated or sham dogs. The results indicate that ROS are critical for the development of the coronary collateral circulation.     

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR.

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.     

Reactive oxygen species differentially affect T cell receptor-signaling pathways

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.