Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

自由基对线粒体DNA的氧化损伤与衰老

自由基是一类氧化剂,对生物具有多种损害作用．衰老的自由基学说是有关衰老机理的诸多学说之一．线粒体DNA组成结构特殊,易受自由基攻击;目前认为,线粒体DNA的氧化损伤是自由基引起衰老的分子基础．     

Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.     

Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants alpha-tocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by alpha-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by alpha-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators.     

Fatty acid oxidation and isoprostanes: Oxidative strain and oxidative stress

Oxidative stress is implicated as one of the key causes underlying many diseases. Free radicals are important constituents of basal physiology. Assessment of free radicals or the end products of their action has proved to be difficult. Consequently, authentication of the contribution of free radicals to physiology and pathology has usually been equivocal. Isoprostanes are biosynthesized in vivo, predominantly through free radical attack on arachidonic acid and are now regarded as robust biomarkers of oxidative stress in vivo. Isoprostanes are associated with many human diseases, and their concentration is altered over the course of normal human pregnancy, but their (patho)physiological roles have not yet been clearly defined. Measurement of F₂-isoprostanes in body fluids could offer a unique analytical opportunity to study the role of free radicals in physiology and pathophysiology in order to comprehend both oxidative strain and oxidative stress.     

The tandem of free radicals and methylglyoxal

Methylglyoxal is an alpha-oxoaldehyde inevitably produced from triose-phosphate intermediates of phosphorylating glycolysis, and also from amino acids and acetone. Recently, the attention has been focused on the involvement of free radicals in methylglyoxal toxicity. In this review, a summary of the relationship between methylglyoxal metabolism and free radical production is presented, extending discussion from the possible metabolic routes to the toxicological events by reviewing the role of free radicals in both generation and degradation of this 1,2-dicarbonyl as well as in the modification of biological macromolecules, and focusing on the action of methylglyoxal upon cellular glutathione content. Methylglyoxal-provoked free radical generation involving reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as organic radicals like methylglyoxal radial or crosslinked protein radical as potential risk factors to tissue damage propagation, is thoroughly discussed. Special attention is paid to the potential therapeutic interventions. The paper arrives at the conclusion that a tight junction exists between methylglyoxal toxicity and free radical (particularly ROS) generation, though the toxicity of 1,2-dicarbonyl evolves even under anaerobic conditions, too. The events follow a sequence beginning with carbonyl stress essential for the toxicity, leading to free radical formation and finally ending in either apoptosis or necrosis. Both oxidative and nitrosative stress play important but not indispensable role in the development of methylglyoxal toxicity.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

Potential markers of oxidative stress in stroke

Free radical production is increased in ischemic and hemorrhagic stroke, leading to oxidative stress that contributes to brain damage. The measurement of oxidative stress in stroke would be extremely important for a better understanding of its pathophysiology and for identifying subgroups of patients that might receive targeted therapeutic intervention. Since direct measurement of free radicals and oxidized molecules in the brain is difficult in humans, several biological substances have been investigated as potential peripheral markers. Among lipid peroxidation products, malondialdehyde, despite its relevant methodological limitations, is correlated with the size of ischemic stroke and clinical outcome, while F2-isoprostanes appear to be promising, but they have not been adequately evaluated. 8-Hydroxy-2-deoxyguanosine has been extensively investigated as markers of oxidative DNA damage but no study has been done in stroke patients. Also enzymatic and nonenzymatic antioxidants have been proposed as indirect markers. Among them ascorbic acid, alpha-tocopherol, uric acid, and superoxide dismutase are related to brain damage and clinical outcome. After a critical evaluation of the literature, we conclude that, while an ideal biomarker is not yet available, the balance between antioxidants and by-products of oxidative stress in the organism might be the best approach for the evaluation of oxidative stress in stroke patients.     

Death-associated protein 3 regulates cellular senescence through oxidative stress response

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.     

Hydroxyl radical in living systems and its separation methods

It has recently been shown that hydroxyl radicals are generated under physiological and pathological conditions and that they seem to be closely linked to various models of pathology putatively implying oxidative stress. It is now recognized that the hydroxyl radical is well-regulated to help maintain homeostasis on the cellular level in normal, healthy tissues. Conversely, it is also known that virtually every disease state involves free radicals, particularly the most reactive hydroxyl radical. However, when hydroxyl radicals are generated in excess or the cellular antioxidant defense is deficient, they can stimulate free radical chain reactions by interacting with proteins, lipids, and nucleic acids causing cellular damage and even diseases. Therefore, a confident analytical approach is needed to ascertain the importance of hydroxyl radicals in biological systems. In this paper, we provide information on hydroxyl radical trapping and detection methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence. In addition, the relationships between diseases and the hydroxyl radical in living systems, as well as novel separation methods for the hydroxyl radical are discussed in this paper.     

Acrolein scavenging: a potential novel mechanism of attenuating oxidative stress following spinal cord injury

It has long been established that oxidative stress plays a critical role in the pathophysiology of spinal cord injury, and represents an important target of therapeutic intervention following the initial trauma. However, free radical scavengers have been largely ineffective in clinical trials, and as such a novel target to attenuate oxidative stress is highly warranted. In addition to free radicals, peroxidation of lipid membranes following spinal cord injury (SCI) produces reactive aldehydes such as acrolein. Acrolein is capable of depleting endogenous antioxidants such as glutathione, generating free radicals, promoting oxidative stress, and damaging proteins and DNA. Acrolein has a significantly longer half‐life than the transient free radicals, and thus may represent a potentially better target of therapeutic intervention to attenuate oxidative stress. There is growing evidence, from our lab and others, to suggest that reactive aldehydes such as acrolein play a critical role in oxidative stress and SCI. The focus of this review is to summarize the cellular and biochemical mechanisms of acrolein‐induced membrane damage, mitochondrial injury, oxidative stress, cell death, and functional loss. Evidence will also be presented to suggest that acrolein scavenging may be a novel means of therapeutic intervention to attenuate oxidative stress and improve recovery following traumatic SCI.     

Implication of free radical mechanisms in ethanol-induced cellular injury.

Numerous experimental data reviewed in the present article indicate that free radical mechanisms contribute to ethanol-induced liver injury. Increased generation of oxygen- and ethanol-derived free radicals has been observed at the microsomal level, especially through the intervention of the ethanol-inducible cytochrome P450 isoform (CYP2E1). Furthermore, an ethanol-linked enhancement in free radical generation can occur through the cytosolic xanthine and/or aldehyde oxidases, as well as through the mitochondrial respiratory chain. Ethanol administration also elicits hepatic disturbances in the availability of non-safely-sequestered iron derivatives and in the antioxidant defense. The resulting oxidative stress leads, in some experimental conditions, to enhanced lipid peroxidation and can also affect other important cellular components, such as proteins or DNA. The reported production of a chemoattractant for human neutrophils may be of special importance in the pathogenesis of alcoholic hepatitis. Free radical mechanisms also appear to be implicated in the toxicity of ethanol on various extrahepatic tissues. Most of the experimental data available concern the gastric mucosa, the central nervous system, the heart, and the testes. Clinical studies have not yet demonstrated the role of free radical mechanisms in the pathogenesis of ethanol-induced cellular injury in alcoholics. However, many data support the involvement of such mechanisms and suggest that dietary and/or pharmacological agents able to prevent an ethanol-induced oxidative stress may reduce the incidence of ethanol toxicity in humans.     

A review of the molecular mechanisms of hyperglycemia-induced free radical generation leading to oxidative stress

The prevalence of diabetes is growing worldwide with an increasing morbidity and mortality associated with the development of diabetes complications. Free radical production is a normal biological process that is strictly controlled and has been shown to be important in normal cellular homeostasis, and in the bodies response to pathogens. However, there are several mechanisms leading to excessive free radical production that overcome the normal protective quenching mechanisms. Studies have shown that many of the diabetes complications result from excessive free radical generation and oxidative stress, and it has been shown that chronic hyperglycemia is a potent inducer for free radical production, generated through several pathways and triggering multiple molecular mechanisms. An understanding of these processes may help us to improving our preventive or therapeutic strategies. In this review, the major molecular pathways involved in free radical generation induced by hyperglycemia are described.     

Organismal Aging and Oxidants beyond Macromolecules Damage

The relationship between oxidants and organismal aging was first articulated through the free radical theory of aging. One of the major predictions of the free radical theory of aging is that oxidative stress shortens organisms’ lifespan because of an increased level of oxidants, which are damaging to macromolecules. However, challenging the role of oxidants in age‐related diseases, there is now sufficient evidence that antioxidant supplements do not provide significant health benefits. Interestingly, in addition to an increase in oxidant‐mediated macromolecules damage, there is convincing experimental data to support the role of senescent cells in the process of aging. Here, the current knowledge regarding the role of oxidants and cellular senescence in organismal aging is reviewed and it is proposed that, in addition to the role of oxidants as inducers of macromolecular damage, oxidants may also function as regulators of signaling pathways involved in the establishment of cellular senescence. If this role for oxidants is established, it may be necessary to modify the free radical theory of aging from “Organisms age because cells accumulate reactive oxygen species‐dependent damage over time” to: “Organisms age because cells accumulate oxidants’‐dependent damage and oxidants’‐dependent senescent characteristics over time.”     

The tumor suppressor gene ARF as a sensor of oxidative stress

Oxidative stress as a result of either exogenous stimuli or cellular metabolism affects several cellular processes such as proliferation, apoptosis, cell death and senescence. Consequently, it is implicated in the pathogenesis of various human diseases like cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases and aging. Oxidative stress is implicated in carcinogenesis either by directly provoking DNA damage or through the regulation of intracellular signaling cascades. In both cases the cellular response to oxidative stress is determined by the cellular context. ARF, the alternative protein product of the CDKN2A locus has been recently recognized as a novel sensor of oxidative stress, in a β-catenin and Hsp70-mediated manner. Since, improved understanding of cellular responses to oxidative stress may facilitate the design of novel antineoplastic regimens, we herein review the mechanisms by which oxidative stress promotes carcinogenesis, focusing on the role of ARF as a sensor of oxidative stress.     

STING promotes senescence,apoptosis, and extracellular matrix degradation in osteoarthritis via the NF-κB signaling pathway

Damaged deoxyribonucleic acid (DNA) is a primary pathologic factor for osteoarthritis (OA); however, the mechanism by which DNA damage drives OA is unclear. Previous research demonstrated that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) participates in DNA damage response. As a result, the current study aimed at exploring the role STING, which is the major effector in the cGAS-STING signaling casacde, in OA progress in vitro, as well as in vivo. In this study, the expression of STING was evaluated in the human and mouse OA tissues, and in chondrocytes exposed to interleukin-1 beta (IL-1β). The influences of STING on the metabolism of the extracellular matrix (ECM), apoptosis, and senescence, were assessed in STING overexpressing and knocking-down chondrocytes. Moreover, the NF-κB-signaling casacde and its role in the regulatory effects of STING on ECM metabolism, apoptosis, and senescence were explored. The STING knockdown lentivirus was intra-articularly injected to evaluate its therapeutic impact on OA in mice in vivo. The results showed that the expression of STING was remarkably elevated in the human and mouse OA tissues and in chondrocytes exposed to IL-1β. Overexpression of STING promoted the expression of MMP13, as well as ADAMTS5, but suppressed the expression of Aggrecan, as well as Collagen II; it also enhanced apoptosis and senescence in chondrocytes exposed to and those untreated with IL-1β. The mechanistic study showed that STING activated NF-κB signaling cascade, whereas the blockage of NF-κB signaling attenuated STING-induced apoptosis and senescence, and ameliorated STING-induced ECM metabolism imbalance. In in vivo study, it was demonstrated that STING knockdown alleviated destabilization of the medial meniscus-induced OA development in mice. In conclusion, STING promotes OA by activating the NF-κB signaling cascade, whereas suppression of STING may provide a novel approach for OA therapy.Subject terms: Apoptosis, Senescence     

Free radicals in the 1900's: from in vitro to in vivo

Remarkable progress has been achieved in the past 100 years in the field of free radical chemistry, biology and medicine since the discovery of free radicals in 1900. Free radical-mediated processes play a major role in the present industrial chemistry, but they also cause deleterious effects on rubber, plastics, oil products and foods. The importance of free radicals in vivo has been recognized increasingly from both positive and negative sides. Free radicals play an important role in phagocytosis, the production of some biologically essential compounds and possibly cell signaling. At the same time, they may cause oxidative modification of biological molecules, which leads to oxidative damage and eventually to various diseases, cancer and aging. The role and beneficial effects of antioxidants against such oxidative stress support this view. Furthermore, novel issues have been continuously found in this fascinating and yet controversial field of free radicals in biology. In this short article, the past work, present problems and future perspectives of free radicals in life science will be briefly discussed.