Blood-brain barrier (BBB) breakdown is one of the pathophysiological characteristics of ischemic stroke, which may contribute to the progression of brain tissue damage and subsequent neurological impairment. Human immunodeficiency virus (HIV)-infected individuals are at greater risk for ischemic stroke due to diminished immune function and HIV-associated vasculopathy. Studies have shown that astrocytes are involved in maintaining BBB integrity and facilitating HIV-1 infection in the brain. The present study investigated whether targeting astrocyte-endothelial cell signaling with cenicriviroc (CVC), a dual chemokine receptor (CCR)2 and CCR5 antagonist, may protect against dysregulation of cross talk between these cells after oxygen-glucose deprivation/reoxygenation (OGD/R) combined with HIV-1 infection. Permeability assay with 10 kDa fluorescein isothiocyanate (FITC)-dextran demonstrated that CVC alleviated endothelial barrier disruption in noncontact coculture of human brain microvascular endothelial cells (HBMECs) with HIV-1-infected human astrocytes, and reversed downregulation of tight junction protein claudin-5 induced by OGD/R- and HIV-1. Moreover, CVC attenuated OGD/R- and HIV-1-triggered upregulation of the NOD-like receptor protein-3 (NLRP3) inflammasome and IL-1β secretion. Treatment with CVC also suppressed astrocyte pyroptosis by attenuating cleaved caspase-1 levels and the formation of cleaved N-terminal GSDMD (N-GSDMD). Secretome profiling revealed that CVC ameliorated secretion levels of chemokine CC chemokine ligand 17 (CCL17), adhesion molecule intercellular adhesion molecule-1 (ICAM-1), and T cell activation modulator T cell immunoglobulin and mucin domain 3 (TIM-3) by astrocytes synergistically induced by OGD/R and HIV-1. Overall, these results suggest that CVC contributes to restoring astrocyte-endothelial cross interactions in an astrocyte-dependent manner via protection against NLRP3 activation and pyroptosis.NEW & NOTEWORTHY The present study reveals the role of astrocytic NOD-like receptor protein-3 (NLRP3) inflammasome in dysfunctional astrocyte-endothelial cross interactions triggered in response to oxygen/glucose deprivation injury associated with human immunodeficiency virus type 1 (HIV-1) infection. Our results suggest that blocking NLRP3 inflammasome activation and pyroptosis-mediated inflammation with cenicriviroc (CVC) may constitute a potentially effective therapeutic strategy for blood-brain barrier (BBB) protection during HIV-1-associated ischemic stroke.

Activated effector T cells (Teff) and/or compromised regulatory T cells (Treg) underlie many chronic inflammatory diseases. We discovered a novel pathway to regulate survival and expansion of Teff without compromising Treg survival and a potential therapeutic to treat these diseases. We found dimethylguanidino valeric acid (DMGV) as a rheostat for Teff survival: while cell-intrinsic DMGV generated by Alanine-Glyoxylate Aminotransferase 2 (AGXT2) is essential for survival and expansion by inducing mitochondrial ROS and regulation of glycolysis, an excessive (or exogenous) DMGV level inhibits activated Teff survival, thereby the AGXT2-DMGV-ROS axis functioning as a switch to turn on and off Teff expansion. DMGV-induced ROS is essential for glycolysis in Teff, and paradoxically DMGV induces ROS only when glycolysis is active. Mechanistically, DMGV rapidly activates mitochondrial calcium uniporter (MCU), causing a surge in mitochondrial Ca2+ without provoking calcium influx to the cytosol. The mitochondrial Ca2+ surge in turn triggers the mitochondrial Na+/Ca2+ exchanger (NCLX) and the subsequent mitochondrial Na+ import induces ROS by uncoupling the Coenzyme Q cycle in Complex III of the electron transport chain. In preclinical studies, DMGV administration significantly diminished the number of inflammatory T cells, effectively suppressing chronic inflammation in mouse models of colitis and rheumatoid arthritis. DMGV also suppressed expansion of cancer cells in vitro and in a mouse T cell leukemic model by the same mechanism. Our data provide a new pathway regulating T cell survival and a novel mode to treat autoimmune diseases and cancers.

Hypochlorite (HOCl) is one of the most important mediators of inflammatory processes. Recent evidence demonstrates that changes in intracellular calcium pool play a significant role in the damaging effects of hypochlorite and other oxidants. Mitochondria are shown to be one of the intracellular targets of hypochlorite. But little is known about the mitochondrial calcium pool changes in HOCl-induced mitochondrial dysfunction. Using isolated rat liver mitochondria, we showed the oxidative damage of mitochondria (GSH oxidation and mixed protein-glutathione formation without membrane lipid peroxidation) and alterations in the mitochondrial functional parameters (decrease of respiratory activity and efficiency of oxidative phosphorylation, NADH and FADH coenzyme levels, and membrane potential) under hypochlorite action (50-300 μM). Simultaneously, the mitochondrial calcium release and swelling were demonstrated. In the presence of EGTA, the damaging effects of HOCl were less pronounced, reflecting direct involvement of mitochondrial Ca²⁺ in mechanisms of oxidant-induced injury. Furthermore, exposure of HeLa cells to hypochlorite resulted in a considerable increase in cytoplasmic calcium concentrations and a decrease in mitochondrial ones. Applying specific inhibitors of calcium transfer systems, we demonstrated that mitochondria play a key role in the redistribution of cytoplasmic Ca²⁺ ions under hypochlorite action and act as mediators of calcium release from the endoplasmic reticulum into the cytoplasm.

Heart failure (HF) is a progressive, debilitating condition characterized, in part, by altered ionic equilibria, increased ROS production and impaired cellular energy metabolism, contributing to variable profiles of systolic and diastolic dysfunction with significant functional limitations and risk of premature death. We summarize current knowledge concerning changes of intracellular Na+ and Ca2+ control mechanisms during the disease progression and their consequences on mitochondrial Ca2+ homeostasis and the shift in redox balance. Absent existing biological data, our computational modeling studies advance a new 'in silico' analysis to reconcile existing opposing views, based on different experimental HF models, regarding variations in mitochondrial Ca2+ concentration that participate in triggering and perpetuating oxidative stress in the failing heart and their impact on cardiac energetics. In agreement with our hypothesis and the literature, model simulations demonstrate the possibility that the heart's redox status together with cytoplasmic Na+ concentrations act as regulators of mitochondrial Ca2+ levels in HF and of the bioenergetics response that will ultimately drive ATP supply and oxidative stress. The resulting model predictions propose future directions to study the evolution of HF as well as other types of heart disease, and to develop novel testable mechanistic hypotheses that may lead to improved therapeutics.

Stroke is the second leading cause of death and the leading cause of long-term disability in the world. There is an urgent unmet need to develop a range of neuroprotective strategies to restrain the damage that occurs in the hours and days following a stroke. Storax, a natural resin extracted from injuring Liquidambar orientalis Mill, has been used to treat acute stroke in traditional Chinese medicine for many centuries. Storax has demonstrated the neuroprotective effects in cerebrovascular diseases. However, the neuroprotective mechanisms activated by storax in ischemia/reperfusion-injured astrocytes have not been elucidated. In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytes injury model to investigate the effects of storax on OGD/R-induced astrocytes injury and potential mechanisms. Experimental results showed that storax alleviated expression of inflammatory cytokines and protected primary cortical astrocytes injured by OGD/R. Furthermore, storax could inhibit NF-κB activation in injured astrocytes by OGD/R and inhibition of NF-κB with Bay-11-7082 obscured the neuroprotective effects of storax. In conclusion, storax alleviated expression of inflammatory cytokines and protected primary cortical astrocytes injured by OGD/R, which was partially mediated by NF-κB signaling pathway activation.

Until recently, astrocyte processes were thought to be too small to contain mitochondria. However, it is now clear that mitochondria are found throughout fine astrocyte processes and are mobile with neuronal activity resulting in positioning near synapses. In this review, we discuss evidence that astrocytic mitochondria confer selective resiliency to astrocytes during ischemic insults and the functional significance of these mitochondria for normal brain function.

The free-radical theory of aging was proposed more than 50 years ago. As one of the most popular mechanisms explaining the aging process, it has been extensively studied in several model organisms. However, the results remain controversial. The mitochondrial version of free-radical theory of aging proposes that mitochondria are both the primary sources of reactive oxygen species (ROS) and the primary targets of ROS-induced damage. One critical ROS is hydrogen peroxide, which is naturally degraded by catalase in peroxisomes or glutathione peroxidase within mitochondria. Our laboratory developed mice-overexpressing catalase targeted to mitochondria (mCAT), peroxisomes (pCAT), or the nucleus (nCAT) in order to investigate the role of hydrogen peroxide in different subcellular compartments in aging and age-related diseases. The mCAT mice have demonstrated the largest effects on life span and healthspan extension. This chapter will discuss the mCAT phenotype and review studies using mCAT to investigate the roles of mitochondrial oxidative stresses in various disease models, including metabolic syndrome and atherosclerosis, cardiac aging, heart failure, skeletal muscle pathology, sensory defect, neurodegenerative diseases, and cancer. As ROS has been increasingly recognized as essential signaling molecules that may be beneficial in hormesis, stress response and immunity, the potential pleiotropic, or adverse effects of mCAT are also discussed. Finally, the development of small-molecule mitochondrial-targeted therapeutic approaches is reviewed.

Recently, we demonstrated that different running overtraining (OT) protocols with the same external load, but performed downhill (OTR/down), uphill (OTR/up), and without inclination (OTR), led to hepatic fat accumulation. As the disruption of endoplasmic reticulum (ER) homeostasis is linked to animal models of fatty liver disease, we investigated the effects of these OT models on the proteins related to ER stress (i.e., BiP, inositol-requiring enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, eIF2alpha, ATF6beta, and glucose-regulated protein 94) and apoptosis (C/EBP-homologous protein, Caspase-3, 4, and 12, Bax, and tumor necrosis factor receptor-associated factor 2) in livers of C57BL/6 mice. Also, aerobic training can attenuate cardiac ER stress and improve exercise capacity. Therefore, we investigated whether the decrease in performance induced by our OT protocols is linked to ER stress and apoptosis in mouse hearts. The rodents were divided into six groups: naïve (N, sedentary mice), control (CT, sedentary mice submitted to the performance evaluations), trained (TR), OTR/down, OTR/up, and OTR groups. Rotarod, incremental load, exhaustive, and grip force tests were used to evaluate performance. After the grip force test, the livers and cardiac muscles (i.e., left ventricle) were removed and used for immunoblotting. All of the OT protocols led to similar responses in the performance parameters and displayed significantly lower hepatic ATF6beta values compared to the N group. The OTR/down group exhibited lower liver cleaved caspase-3 values compared to the CT group. However, the other proteins related to ER stress and apoptosis were not modulated. Also, the cardiac proteins related to ER stress and apoptosis were not modulated in the experimental groups. In conclusion, the OT protocols decreased the levels of hepatic ATF6beta, and the OTR/down group decreased the levels of hepatic cleaved caspase-3. Also, the decrease in performance induced by our OT models is not associated with ER stress and apoptosis in mice hearts.

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.

The signals and mechanisms that synchronize the timing of human parturition remain a mystery and a better understanding of these processes is essential to avert adverse pregnancy outcomes. Although our insights into human labor initiation have been informed by studies in animal models, the timing of parturition relative to fetal maturation varies among viviparous species, indicative of phylogenetically different clocks and alarms; but what is clear is that important common pathways must converge to control the birth process. For example, in all species, parturition involves the transition of the myometrium from a relaxed to a highly excitable state, where the muscle rhythmically and forcefully contracts, softening the cervical extracellular matrix to allow distensibility and dilatation and thus a shearing of the fetal membranes to facilitate their rupture. We review a number of theories promulgated to explain how a variety of different timing mechanisms, including fetal membrane cell senescence, circadian endocrine clocks, and inflammatory and mechanical factors, are coordinated as initiators and effectors of parturition. Many of these factors have been independently described with a focus on specific tissue compartments.In this review, we put forth the core hypothesis that fetal membrane (amnion and chorion) senescence is the initiator of a coordinated, redundant signal cascade leading to parturition. Whether modified by oxidative stress or other factors, this process constitutes a counting device, i.e. a clock, that measures maturation of the fetal organ systems and the production of hormones and other soluble mediators (including alarmins) and that promotes inflammation and orchestrates an immune cascade to propagate signals across different uterine compartments. This mechanism in turn sensitizes decidual responsiveness and eventually promotes functional progesterone withdrawal in the myometrium, leading to increased myometrial cell contraction and the triggering of parturition. Linkage of these processes allows convergence and integration of the gestational clocks and alarms, prompting a timely and safe birth. In summary, we provide a comprehensive synthesis of the mediators that contribute to the timing of human labor. Integrating these concepts will provide a better understanding of human parturition and ultimately improve pregnancy outcomes.

Naoxintong capsule (NXT), developed from Buyang Huanwu Decoction, has shown the neuroprotective effects in cerebrovascular diseases, but the neuroprotection mechanisms of NXT on ischemia/reperfusion injured neurons have not yet been well known. In this study, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurons injury model and treat the neurons with cerebrospinal fluid containing NXT (BNC) to investigate the effects of NXT on OGD/R induced neurons injury and potential mechanisms. BNC improved neuron viability and decreased apoptotic rate induced by OGD/R. BNC attenuated OGD/R induced cytosolic and mitochondrial Ca(2+) overload, ROS generation, intracellular NO levels and nNOS mRNA increase, and cytochrome-c release when compared with OGD/R group. BNC significantly inhibited both mPTP opening and ΔΨm depolarization. BNC increased Bcl-2 expression and decreased Bax expression, upregulated the Bcl-2/Bax ratio, downregulated caspase-3 mRNA and caspase-9 mRNA expression, and decreased cleaved caspase-3 expression and caspase-3 activity. BNC increased phosphorylation of Akt following OGD/R, while LY294002 attenuated BNC induced increase of phosphorylated Akt expression. Our study demonstrated that NXT protected primary neurons from OGD/R induced injury by inhibiting calcium overload and ROS generation, protecting mitochondria, and inhibiting mitochondrial apoptotic pathway which was mediated partially by PI3K-Akt signaling pathway activation.

Danhong injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been widely used for the treatment of ischemic heart disease, and clinical and experimental studies have demonstrated the protective effects against myocardial ischemia/reperfusion injury. Nevertheless, the underlying cellular mechanisms responsible for this protective effect are poorly understood.

The present study aimed to examine the mechanism of DHI in regulating hypoxia/reoxygenation- and H2O2-induced cardiomyocytes injury.

Neonatal rat cardiomyocytes were subjected to hypoxia (9h)-reoxygenation (2h) or H2O2 (100 μM) in the presence or absence of DHI (2.5, 5, 10 μg/mL). Intracellular reactive oxygen species (ROS), cytosolic and mitochondrial Ca(2+) concentrations, mitochondrial membrane potential (ΔΨm) and mitochondrial permeability transition pore (mPTP) opening were monitored using CMH2DCFDA, Fluo-4 and rhod-2, JC-1 and calcein, respectively. Cell survival was evaluated using the 2-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and apoptosis was detected by Annexin V/propidium iodide (PI) staining.

DHI improved cell survival following H/R and H2O2 injury and reduced H/R-induced cytochrome c release and apoptosis when compared with non-DHI treated cells. In addition, DHI attenuated H/R-induced ROS generation, H2O2-induced cytosolic and mitochondrial Ca(2+) overload, and cellular ROS generation when compared with H/R- and H2O2-only groups. Moreover, DHI significantly inhibited both mPTP opening and ΔΨm depolarization.

These data demonstrate that the protective mechanism of DHI against H/R- and H2O2-induced injury is mediated by the inhibition of mPTP opening via mitigating Ca(2+) overload and ROS generation.

Mitochondria are one of the major sites for the generation of reactive oxygen species (ROS) as an undesirable side product of oxidative energy metabolism. Damaged mitochondria can augment the generation of ROS. Dysfunction of mitochondria increase the risk for a large number of human diseases, including cardiovascular diseases (CVDs). Heart failure (HF) following ischemic heart disease, infantile cardiomyopathy and cardiac hypertrophy associated with left ventricular dilations are some of the CVDs in which the role of mitochondrial oxidative stress has been reported. Advances in mitochondrial research during the last decade focused on the preservation of its function in the myocardium, which is vital for the cellular energy production. Experimental and clinical trials have been conducted using mitochondria-targeted molecules like: MnSOD mimetics, such as EUK-8, EUK-134 and MitoSOD; choline esters of glutathione and N-acetyl-L-cysteine; triphenylphosphonium ligated vitamin E, lipoic acid, plastoquinone and mitoCoQ10; and Szeto-Schiller (SS)- peptides (SS-02 and SS-31). Although many results are inconclusive, some of the findings, especially on CoQ10, are worthwhile. This review summarizes the role of mitochondria-targeted delivery of agents and their consequences in the control of HF.

Endothelial cell dysfunction is the hallmark of every cardiovascular disease/condition, including atherosclerosis and ischemia/reperfusion injury. Fluid shear stress acting on the vascular endothelium is known to regulate cell homeostasis. Altered hemodynamics is thought to play a causative role in endothelial dysfunction. The dysfunction is associated with/preceded by mitochondrial oxidative stress. Studies by our group and others have shown that the form and/or function of the mitochondrial network are affected when endothelial cells are exposed to shear stress in the absence or presence of additional physicochemical stimuli. The present review will summarize the current knowledge on the interconnections among intracellular Ca²⁺ - nitric oxide - mitochondrial reactive oxygen species, mitochondrial fusion/fission, autophagy/mitophagy, and cell apoptosis vs. survival. More specifically, it will list the evidence on potential regulation of the above intracellular species and processes by the fluid shear stress acting on the endothelium under either physiological flow conditions or during reperfusion (following a period of ischemia). Understanding how the local hemodynamics affects mitochondrial physiology and the cell redox state may lead to development of novel therapeutic strategies for prevention or treatment of the endothelial dysfunction and, hence, of cardiovascular disease.

Ischemic stroke exhibits a multiplicity of pathophysiological mechanisms. To address the diverse pathophysiological mechanisms observed in ischemic stroke investigators seek to find therapeutic strategies that are multifaceted in their action by either investigating multipotential compounds or by using a combination of compounds. Taurine, an endogenous amino acid, exhibits a plethora of physiological functions. It exhibits antioxidative properties, stabilizes membrane, functions as an osmoregulator, modulates ionic movements, reduces the level of pro-inflammators, regulates intracellular calcium concentration; all of which contributes to its neuroprotective effect. Data are accumulating that show the neuroprotective mechanisms of taurine against stroke pathophysiology. In this review, we describe the neuroprotective mechanisms employed by taurine against ischemic stroke and its use in clinical trial for ischemic stroke.

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125-1000μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2',7' dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We tested the hypothesis that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response and that exercise training could reverse this phenotype. MPT was assessed by mitochondrial swelling in response to 10-100 μM Ca(2+). FH pigs did show an increased MPT response to Ca(2+) that was associated with decreases in the expression of the putative MPT pore components mitochondrial phosphate carrier (PiC) and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation, as well as decreased levels of reduced GSH in cardiac mitochondria. Expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin-2 (Trx2), and peroxiredoxin-3 (Prx3) was greatly reduced in the FH pigs. In contrast, cytosolic catalase expression and activity were increased. However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce mitochondrial oxidative stress, and return MnSOD, Trx2, Prx3, and catalase expression/activities to normal. We conclude that FH reduces mitochondrial antioxidants, increases mitochondrial oxidative stress, and enhances the MPT response in the porcine myocardium, and that exercise training can reverse these detrimental alterations.

Aseptic loosening is a major cause of failure of total hip arthroplasty (THA). Macrophage apoptosis in interface membrane has been proved to play an important role in the pathogenesis of aseptic loosening. The purpose of current study was to identify the apoptotic mechanism of macrophages in the interface membrane of aseptic loosening. We collected periprosthetic interface membrane from 23 patients undergoing the revision operations for aseptic loosening of hip joint prostheses. To serve as the control group, samples of capsule were collected from 18 patients undergoing the primary hip arthroplasties for osteoarthritis (OA). The ultrastructure of interface membrane was examined by transmission electron microscopy (TEM), and in situ apoptotic macrophage identification was performed by TUNEL staining. Furthermore, using immunohistochemical methods we investigated the expression of some apoptosis-related markers such as inducible nitric oxide synthase (iNOS), peroxynitrite (ONOO(-)), cleaved caspase-3/4/8/9, cytochrome c, glucose regulated protein 78 (GRP78), and growth arrest and DNA damage-inducible gene 153 (GADD153) in macrophages. These markers were regarded as apoptotic inducers or specific indicators of different apoptotic pathways such as death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway. TEM showed that a great deal of wear debris was phagocytosed by macrophages, which displayed morphological changes characteristic of apoptosis. The results of TUNEL staining demonstrated that there were more apoptotic macrophages in interface membrane. The expression levels of iNOS, ONOO(-), cleaved caspase-3/4/8/9, cytochrome c, GRP78 and GADD153 in macrophages in interface membrane were significantly higher than those in the control samples (p < 0.05). Our results suggest that death receptor pathway, mitochondria/cytochrosome c caspase-dependent pathway and ER stress pathway are involved in the process of macrophage apoptosis. A therapeutic target to modulate the apoptotic pathways in macrophages may be a strategy to prevent and treat aseptic loosening.

Cell survival and death are complex matters. Too much survival may lead to cancer and too much cell death may result in tissue degeneration. In this chapter, we will first of all focus on the cellular survival mechanisms that promote correct folding and maintenance of protein function. These mechanisms include protein quality control (PQC) systems comprising molecular chaperones and intracellular proteases in the cytosol, endoplasmatic reticulum (ER) and in the mitochondria. In addition to the PQC systems, mechanisms elicited by misfolded proteins, known as unfolded protein responses (UPRs), including induction/activation of antioxidant systems are also present in the three compartments of the cell. Second, we will discuss the mechanisms by which misfolded proteins lead to the generation of oxidative stress in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These species are produced mainly from superoxide (O2-) generated in the mitochondrial respiratory chain and from nitrogen oxide (NO) produced by the mitochondrial nitrogen oxide synthetase (mtNOS). Third, the effects of oxidative stress will be discussed, both with respect to mitochondrial dynamics, i.e., fission and fusion, and the related elimination of dysfunctional mitochondria by cellular cleaning systems, i.e., mitophagy or mitoptosis, and related to the generation and cellular effects of oxidatively modified proteins, which closes a vicious cycle of protein misfolding and oxidative stress.

Mitochondrial function is integral to maintaining cellular homeostasis through the production of ATP, the generation of reactive oxygen species (ROS) for signaling, and the regulation of the apoptotic cascade. A number of small molecules, including nitric oxide (NO), are well-characterized regulators of mitochondrial function. Nitrite, an NO metabolite, has recently been described as an endocrine reserve of NO that is reduced to bioavailable NO during hypoxia to mediate physiological responses. Accumulating data suggests that mitochondria may play a role in metabolizing nitrite and that nitrite is a regulator of mitochondrial function. Here, what is known about the interactions of nitrite with the mitochondria is reviewed, with a focus on the role of the mitochondrion as a metabolizer and target of nitrite.

Stroke is the third leading cause of death in the United States, yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules that mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoPs), formed after free radical-mediated peroxidation of arachidonic acid, are used as markers of stress, but their bioactivity is poorly understood. We have recently shown that 15-A(2t)-IsoP is a potent neurotoxin in vitro and increases the free radical burden in neurons. In this work, we demonstrate that 15-A(2t)-IsoP is abundantly produced in stroke-infarcted human cortical tissue. Using primary neuronal cultures we found that minimally toxic exposure to 15-A(2t)-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation resulted in a significant hyperpolarization of the mitochondrial membrane and dramatically increased neuronal cell death. In the presence of Ca(2+), 15-A(2t)-IsoP led to a rapid induction of the permeability transition pore and release of cytochrome c. Taken with our previous work, these data support a model in which ischemia causes generation of reactive oxygen species, calcium influx, lipid peroxidation, and 15-A(2t)-IsoP formation. These factors combine to enhance opening of the permeability transition pore leading to cell death subsequent to mitochondrial cytochrome c release. These data are the first documentation of significant 15-A(2t)-IsoP formation after acute ischemic stroke and suggest that the addition of 15-A(2t)-IsoP to in vitro models of ischemia may help to more fully recapitulate stroke injury.

Inhibition of Na+/H+ exchange (NHE) during ischemia reduces cardiac injury due to reduced reverse mode Na+/Ca2+ exchange. We hypothesized that activating NHE-1 at buffer pH 8 during ischemia increases mitochondrial oxidation, Ca2+ overload, and reactive O2 species (ROS) levels and worsens functional recovery in isolated hearts and that NHE inhibition reverses these effects. Guinea pig hearts were perfused with buffer at pH 7.4 (control) or pH 8 +/- NHE inhibitor eniporide for 10 minutes before and for 10 minutes after 35- minute ischemia and then for 110 minutes with pH 7.4 buffer alone. Mitochondrial NADH and FAD, [Ca2+], and superoxide were measured by spectrophotofluorometry. NADH and FAD were more oxidized, and cardiac function was worse throughout reperfusion after pH 8 versus pH 7.4, Ca2+ overload was greater at 10-minute reperfusion, and superoxide generation was higher at 30-minute reperfusion. The pH 7.4 and eniporide groups exhibited similar mitochondrial function, and cardiac performance was most improved after pH 7.4+eniporide. Cardiac function on reperfusion after pH 8+eniporide was better than after pH 8. Percent infarction was largest after pH 8 and smallest after pH 7.4+eniporide. Activation of NHE with pH 8 buffer and the subsequent decline in redox state with greater ROS and Ca2+ loading underlie the poor functional recovery after ischemia and reperfusion.

During cardiac ischemia-reperfusion (IR) injury, excessive generation of reactive oxygen species (ROS) and overload of Ca(2+) at the mitochondrial level both lead to opening of the mitochondrial permeability transition (PT) pore on reperfusion. This can result in the depletion of ATP, irreversible oxidation of proteins, lipids, and DNA within the cardiomyocyte, and can trigger cell-death pathways. In contrast, mitochondria are also implicated in the cardioprotective signaling processes of ischemic preconditioning (IPC), to prevent IR-related pathology. Nitric oxide (NO*) has emerged as a potent effector molecule for a variety of cardioprotective strategies, including IPC. Whereas NO* is most noted for its activation of the "classic" soluble guanylate cyclase (sGC) signaling pathway, emerging evidence indicates that NO can directly act on mitochondria, independent of the sGC pathway, affording acute cardioprotection against IR injury. These direct effects of NO* on mitochondria are the focus of this review.

The endoplasmic reticulum (ER) is a well-orchestrated protein-folding machine composed of protein chaperones, proteins that catalyze protein folding, and sensors that detect the presence of misfolded or unfolded proteins. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed toward a degradative pathway. The unfolded protein response (UPR) is an intracellular signaling pathway that coordinates ER protein-folding demand with protein-folding capacity and is essential to adapt to homeostatic alterations that cause protein misfolding. These include changes in intraluminal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. The ER provides a unique oxidizing folding-environment that favors the formation of the disulfide bonds. Accumulating evidence suggests that protein folding and generation of reactive oxygen species (ROS) as a byproduct of protein oxidation in the ER are closely linked events. It has also become apparent that activation of the UPR on exposure to oxidative stress is an adaptive mechanism to preserve cell function and survival. Persistent oxidative stress and protein misfolding initiate apoptotic cascades and are now known to play predominant roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis, and neurodegenerative diseases.

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.

Damage to heart mitochondrial structure and function occur with aging, and in heart failure (HF). However, the extent of mitochondrial dysfunction, the expression of mitochondrial and nuclear genes, and their cross-talk is not known.

Several observations have suggested that somatic mutations in mitochondrial DNA (mtDNA), induced by reactive oxygen species (ROS), appear to be the primary cause of energy decline, and that the generation of ROS is mainly the product of the mitochondrial respiratory chain. The free radical theory of aging, that could also be applied to HF, and in particular the targeting of mtDNA is supported by a plurality of observations from both animal and clinical studies showing decreased mitochondrial function, increased ROS levels and mtDNA mutations in the aging heart.

Aging and HF with their increased ROS-induced defects in mtDNA, including base modifications and frequency of mtDNA deletions, might be expected to cause increased errors or mutations in mtDNA-encoded enzyme subunits, resulting in impaired oxidative phosphorylation and defective electron transport chain (ETC) activity which in turn creates more ROS. These events in both the aging and failing heart involve substantial nuclear-mitochondrial interaction, which is further illustrated in the progression of myocardial apoptosis. In this review the cross-talk between the nucleus and the mitochondrial organelle will be examined based on a number of animal and clinical studies, including our own.

Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

Mitochondria can serve as the arbiter of cell fate in response to stress. Mitochondrial permeability transition (MPT) is characterized by permeabilization of an otherwise relatively impermeable mitochondrial inner membrane and appears to have a major role in ischemia/reperfusion (I/R) injury in myocardial infarction and stroke. After I/R, the fate of the cell is determined by the extent of MPT. If minimal, the cell may recover; if moderate, the cell may undergo programmed cell death; if severe, the cell may die from necrosis due to inadequate energy production. After reviewing the role of MPT in disease, we examine the signaling and metabolic networks that regulate MPT. We then conclude with some of the challenges in future MPT research.

N-Methyl-D-aspartate-mediated neurotoxicity is known to involve nitric oxide production and to be augmented in an environment of reactive oxygen species. We used TUNEL staining and homogenous cytosolic immunoreactivity of cytochrome c in an acute brain slice preparation to investigate the influence of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, on N-methyl-D-aspartate-induced apoptosis. Cerebrocortical slices were obtained from parietal lobes of 7-day-old Sprague-Dawley rats, superfused with well-oxygenated artificial cerebrospinal fluid, and metabolically recovered. Subsequent 30-min exposures to 10 microM N-methyl-D-aspartate in treated and untreated slices were followed by 4 h of recovery superfusion with oxygenated artificial cerebrospinal fluid. Outcomes were compared for three groups of slices: "the N-methyl-D-aspartate-only group"; "the edaravone treatment group", which had 20 microM edaravone present throughout and subsequent to N-methyl-D-aspartate exposure; the "control group", in which slices were superfused only with oxygenated artificial cerebrospinal fluid. At the conclusion of recovery (t = 4 h), the percentage of TUNEL-positive cells in the edaravone treatment group (7.0+/-3.3%) was significantly reduced from the percentage for the N-methyl-D-aspartate-only group (21.9+/-4.1%), and insignificantly greater than the percentage for the control group (3.4+/-2.1%). Percentages of cytochrome c positive cells at t = 1 h were significantly increased (p < 0.01) in the N-methyl-d-aspartate-only group (30.6+/-1.9%) compared to percentages for both the control group (11.4+/-2.6%) and the edaravone treatment group (15.2+/-2.1%). Edaravone's reduction in TUNEL staining and cytochrome c release provides evidence of reactive oxygen species mechanisms and antioxidant benefits in cytochrome c-mediated apoptosis during N-methyl-D-aspartate excitotoxicity.

Peroxynitrite does not directly cause strand scission of genomic DNA. Rather, as we previously reported, the DNA cleavage is largely mediated by H(2)O(2) resulting from the dismutation of superoxide generated in the mitochondria upon peroxynitrite-dependent inhibition of complex III. The present study demonstrates that this process is strictly controlled by the availability of Ca(2+) in the mitochondrial compartment. Experiments using intact as well as permeabilized U937 cells showed that the DNA-damaging response evoked by peroxynitrite is enhanced by treatments causing an increase in mitochondrial Ca(2+) uptake and remarkably reduced under conditions leading to inhibition of mitochondrial Ca(2+) accumulation. An additional, important observation was that the source of the Ca(2+) mobilized by peroxynitrite is the ryanodine receptor; preventing the mobilization of Ca(2+) with ryanodine suppressed the mitochondrial formation of reactive oxygen species and the ensuing DNA strand scission. Identical results were obtained using PC12, C6, and THP-1 cells. These results, along with our previous findings indicating that the DNA damage induced by peroxynitrite is also suppressed by inhibition of the electron flow through complex I, e.g., by rotenone, or by the respiration-deficient phenotype, demonstrate that the mitochondrial formation of DNA-damaging species is critically regulated by the inhibition of complex III and by the availability of Ca(2+).

The electrophilic lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is known to allow adaptation to oxidative stress in cells at low concentrations and apoptosis at high levels. The mechanisms leading to adaptation involve the covalent modification of regulatory proteins, such as Keap1, and augmentation of antioxidant defences in the cell. The targets leading to apoptosis are less well defined, but mitochondria have been indirectly implicated in the mechanisms of cell death mediated by electrophilic lipids. To determine the potential of electrophilic cyclopentenones to induce pro-apoptotic effects in the mitochondrion, we used isolated liver mitochondria and demonstrated that 15d-PGJ2 promotes Ca2+-induced mitochondrial swelling and cytochrome c release. The mechanisms involved are consistent with direct modification of protein thiols in the mitochondrion, rather than secondary formation of reactive oxygen species or lipid peroxidation. Using proteomic analysis in combination with biotinylated 15d-PGJ2, we were able to identify 17 potential targets of the electrophile-responsive proteome in isolated liver mitochondria. Taken together, these results suggest that electrophilic lipid oxidation products can target a sub-proteome in mitochondria, and this in turn results in the transduction of the electrophilic stimulus to the cell through cytochrome c release.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

Nitric oxide (NO) derived from inducible NO synthase has been implicated in cardiac rejection. However, little is known about the role of the reactive nitrogen species peroxynitrite. We examined the protective actions of a peroxynitrite decomposition catalyst, WW85, in an experimental model of acute cardiac rejection. Heterotopic, abdominal transplantation of rat donor hearts was performed. Groups included isografts, allografts, or allografts treated with WW85, cyclosporine, or cyclosporine + WW85. We determined graft survival, histological rejection, and graft function (by in situ sonomicrometry). Intragraft biochemical analysis of cytokines and proapoptotic and antiapoptotic gene expression using reverse transcriptase-polymerase chain reaction were determined. Treatment with WW85 or cyclosporine alone prolonged graft survival, improved graft function, and decreased histological rejection. Graft survival was further significantly (P < 0.001) enhanced by combination treatment. A decrease was also shown in nitrotyrosine, poly(ADP-ribose) polymerase (PARP) activation, and lipid peroxide formation by WW85 that was potentiated when given in combination with cyclosporine. Benefits could not be ascribed to changes in intragraft myeloperoxidase activity. Only combination therapy produced significant decreases in inflammatory cytokine gene expression, suggesting that WW85 acted primarily downstream of these stimuli. In general, WW85 had no direct action on expression of the proapoptotic gene, Fas ligand; however, WW85 given alone or with cyclosporine enhanced expression of antiapoptotic genes Bcl-2 and Bcl-xL. Collectively, these findings suggest a protective action of the peroxynitrite decomposition catalyst WW85 on graft rejection that is independent of any action on leukocyte sequestration and cytokine gene expression. Rather, effects seem to be downstream on decreased protein nitration, decreased lipid peroxidation, and decreased PARP activation.

Liver cirrhosis is often preceded by overt signs of hepatitis, including parenchymal cell inflammation and infiltration of polymorphonuclear (PMN) leukocytes. Activated PMNs release both reactive oxygen species and reactive halogen species, including hypochlorous acid (HOCl), which are known to be significantly cytotoxic due to their oxidizing potential. Because the role of mitochondria in the hepatotoxicity attributed to HOCl has not been elucidated, we investigated the effects of HOCl on mitochondrial function in the human hepatoma HepG2 cell line, human fetal liver cells, and isolated rat liver mitochondria. We show here that HOCl induced mitochondrial dysfunction, and apoptosis was dependent on the induction of the mitochondrial permeability transition (MPT), because HOCl induced mitochondrial swelling and collapse of the mitochondrial membrane potential with the concomitant release of cytochrome c. These biochemical events were inhibited by the classical MPT inhibitor cyclosporin A (CSA). Cell death induced by HOCl exhibited several classical hallmarks of apoptosis, including annexin V labeling, caspase activation, chromatin condensation, and cell body shrinkage. The induction of apoptosis by HOCl was further supported by the finding that CSA and caspase inhibitors prevented cell death. For the first time, these results show that HOCl activates the MPT, which leads to the induction of apoptosis and provides a novel insight into the mechanisms of HOCl-mediated cell death at sites of chronic inflammation.

During the past decade, enormous advances have been made in cell biology. Major advances included the publication of the human genome sequence, the development of proteomics, and DNA microarray technologies and techniques to selectively "silence" genes using short strands of double-stranded RNA. Some areas of great progress that are particularly relevant to critical care medicine include huge improvements in our understanding of the signal transduction pathways involved in the innate immune response and adaptation to hypoxia. Other areas of important progress include improvements in our understanding of how inflammation causes derangements in epithelial structure and function and impairs cellular utilization of oxygen.

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.