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.     

Invited review: the mitochondrion in osteoarthritis

In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage promote aging, cell death, and ultimately, functional failure and degeneration. Because articular cartilage chondroyctes are highly glycolytic, mitochondrially mediated pathogenesis has not been previously applied in models for pathogenesis of osteoarthritis (OA), a cartilage degenerative disease that increases markedly in aging. However, chondrocyte mitochondria respire in vitro and they demonstrate swelling and changes in number in situ in the course of OA. Normal chondrocyte mitochondrial function is hypothesized to critically support adenosine triphosphate (ATP) reserves in functional stressed chondrocytes during OA evolution. In this model, disruption of chondrocyte respiration by nitric oxide, a mediator markedly up-regulated in OA cartilage, is centrally involved in chondrocyte functional compromise. Furthermore, mitochondrial dysfunction can mediate several specific pathogenic pathways implicated in OA. These include oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, increased chondrocyte apoptosis, and pathologic cartilage matrix calcification. In addition, the direct, sublethal impairment of chondrocyte mitochondrial ATP synthesis in vitro decreases matrix synthesis and increases matrix calcification ('disease in a dish'). The weight of evidence reviewed herein strongly supports chondrocyte mitochondrial impairment as a mediator of the establishment and progression of OA.     

Icariin protects against iron overload-induced bone loss via suppressing oxidative stress

Iron overload is common in patients with diseases such as hemoglobinopathies, hereditary hemochromatosis or elderly men and postmenopausal women. This disorder is frequently associated with bone loss and recently has been considered as an independent risk factor for osteoporosis. By excess reactive oxygen species (ROS) production through Fenton reaction, iron could induce osteoblast apoptosis, inhibit osteoblast osteogenic differentiation. Moreover, Iron could also promote osteoclasts differentiation and bone absorption. The goal of the study is to investigate whether icariin could reverse iron overload-induced bone loss in vitro and in vivo. Icariin is the major active ingredient of Herba Epimedii and has antioxidant, antiosteoporosis functions. In the current study, we demonstrated that oral administration of icariin significantly prevented bone loss in iron overloaded mice. Icariin could protect against iron overload-induced mitochondrial membrane potential dysfunction and ROS production, promote osteoblast survival and reverse the reduction of Runx2, alkaline phosphatase, and osteopontin expression induced by iron overload. Icariin also inhibited osteoclasts differentiation and function. Moreover, we also found that icariin remarkably reduced iron accumulation in bone marrow, suggesting that icariin has the ability to regulate systemic iron metabolism in vivo. These results indicated that icariin could be a potential natural resource for developing medicines to prevent or treat iron overload-induced osteoporosis.     

Epigenetics of Osteoporosis: Critical Analysis of Epigenetic Epidemiology Studies

Osteoarthritis (OA) is an age-related disease with poorly understood pathogenesis. Recent studies have demonstrated that miRNA might play a key role in OA initiation and development. We reviewed recent publications and elucidated the connection between miRNA and OA cartilage anabolic and catabolic signals, including four signaling pathways: TGF-β/Smads and BMPs signaling, associated with cartilage anabolism; and MAPK and NF-KB signaling, associated with cartilage catabolism. We also explored the relationships with MMP, ADAMTS and NOS (NitricOxide Synthases) families, as well as with the catabolic cytokines IL-1 and TNF-α. The potential role of miRNAs in biological processes such as cartilage degeneration, chondrocyte proliferation, and differentiation is discussed. Collective evidence indicates that miRNAs play a critical role in cartilage degeneration. These findings will aid in understanding the molecular network that governs articular cartilage homeostasis and in to elucidate the role of miRNA in the pathogenesis of OA.     

Elevated hepatic iron: A confounding factor in chronic hepatitis C

Historically, iron overload in the liver has been associated with the genetic disorders hereditary hemochromatosis and thalassemia and with unusual dietary habits. More recently, elevated hepatic iron levels also have been observed in chronic hepatitis C virus (HCV) infection. Iron overload in the liver causes many changes including induction of oxidative stress, damage to lysosomes and mitochondria, altered oxidant defense systems and stimulation of hepatocyte proliferation. Chronic HCV infection causes numerous pathogenic changes in the liver including induction of endoplasmic reticulum stress, the unfolded protein response, oxidative stress, mitochondrial dysfunction and altered growth control. Understanding the molecular and cellular changes that could occur in a liver which has elevated hepatic iron levels and in which HCV replication and gene expression are ongoing has clinical relevance and represents an area of research in need of further investigation.     

The dopamine metabolite aminochrome inhibits mitochondrial complex I and modifies the expression of iron transporters DMT1 and FPN1

Hallmarks of idiopathic and some forms of familial Parkinson’s disease are mitochondrial dysfunction, iron accumulation and oxidative stress in dopaminergic neurons of the substantia nigra. There seems to be a causal link between these three conditions, since mitochondrial dysfunction can give rise to increased electron leak and reactive oxygen species production. In turn, recent evidence indicates that diminished activity of mitochondrial complex I results in decreased Fe–S cluster synthesis and anomalous activation of Iron Regulatory Protein 1. Thus, mitochondrial dysfunction could be a founding event in the process that leads to neuronal death. Here, we present evidence showing that at low micromolar concentrations, the dopamine metabolite aminochrome inhibits complex I and ATP production in SH-SY5Y neuroblastoma cells differentiated into a dopaminergic phenotype. This effect is apparently direct, since it is replicated in isolated mitochondria. Additionally, overnight treatment with aminochrome increased the expression of the iron import transporter divalent metal transporter 1 and decreased the expression of the iron export transporter ferroportin 1. In accordance with these findings, cells treated with aminochrome presented increased iron uptake. These results suggest that aminochrome is an endogenous toxin that inhibits by oxidative modifications mitochondrial complex I and modifies the levels of iron transporters in a way that leads to iron accumulation.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy

Under conditions of iron overload, which are now reaching epidemic proportions worldwide, iron-overload cardiomyopathy is the most important prognostic factor in patient survival. We hypothesize that in iron-overload disorders, iron accumulation in the heart depends on ferrous iron (Fe2+) permeation through the L-type voltage-dependent Ca2+ channel (LVDCC), a promiscuous divalent cation transporter. Iron overload in mice was associated with increased mortality, systolic and diastolic dysfunction, bradycardia, hypotension, increased myocardial fibrosis and elevated oxidative stress. Treatment with LVDCC blockers (CCBs; amlodipine and verapamil) at therapeutic levels inhibited the LVDCC current in cardiomyocytes, attenuated myocardial iron accumulation and oxidative stress, improved survival, prevented hypotension and preserved heart structure and function. Consistent with the role of LVDCCs in myocardial iron uptake, iron-overloaded transgenic mice with cardiac-specific overexpression of the LVDCC alpha1-subunit had twofold higher myocardial iron and oxidative stress levels, as well as greater impairment in cardiac function, compared with littermate controls; LVDCC blockade was again protective. Our results indicate that cardiac LVDCCs are key transporters of iron into cardiomyocytes under iron-overloaded conditions, and potentially represent a new therapeutic target to reduce the cardiovascular burden from iron overload.     

Lung injury after ozone exposure is iron dependent

We tested the hypothesis that oxidative stress and biological effect after ozone (O3) exposure are dependent on changes in iron homeostasis. After O3 exposure, healthy volunteers demonstrated increased lavage concentrations of iron, transferrin, lactoferrin, and ferritin. In normal rats, alterations of iron metabolism after O3 exposure were immediate and preceded the inflammatory influx. To test for participation of this disruption in iron homeostasis in lung injury following O3 inhalation, we exposed Belgrade rats, which are functionally deficient in divalent metal transporter 1 (DMT1) as a means of iron uptake, and controls to O3. Iron homeostasis was disrupted to a greater extent and the extent of injury was greater in Belgrade rats than in control rats. Nonheme iron and ferritin concentrations were higher in human bronchial epithelial (HBE) cells exposed to O3 than in HBE cells exposed to filtered air. Aldehyde generation and IL-8 release by the HBE cells was also elevated following O3 exposure. Human embryonic kidney (HEK 293) cells with elevated expression of a DMT1 construct were exposed to filtered air and O3. With exposure to O3, elevated DMT1 expression diminished oxidative stress (i.e., aldehyde generation) and IL-8 release. We conclude that iron participates critically in the oxidative stress and biological effects after O3 exposure.     

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.     

综述:金属离子代谢平衡失调与阿尔茨海默病早期发病机制

研究表明,脑内金属离子代谢失衡与阿尔茨海默病(AD)有关,但其机理尚需深入探讨．结合本实验室研究结果,作者对金属离子代谢紊乱与氧化应激,金属离子代谢紊乱与β-淀粉样蛋白、转铁蛋白和转铁蛋白受体、铁调节蛋白、二价金属离子转运体以及天然抗氧化剂通过调节金属离子代谢平衡缓解β-淀粉样蛋白的毒性和保护细胞的作用进行探讨．提出：铁、铜等金属离子缺乏可能主要与AD早期关系密切,而铁、铜等金属离子过载可能主要与AD后期损伤关系密切的学术观点．     

The relevance of iron in the pathogenesis of Parkinson's disease

Alterations of iron levels in the brain has been observed and documented in a number of neurodegenerative disorders including Parkinson's disease (PD). The elevated nigral iron levels observed in PD may reflect a dysfunction of brain iron homeostasis. Under normal physiological conditions excess iron can be sequestrated in ferritin and neuromelanin. Alternatively, the excess iron may represent a component of brain iron deposition associated with ageing. The aetiology of idiopathic PD largely remains an enigma. However, intensive investigations have provided a host of putative mechanisms that might contribute to the pathogenesis underlying the characteristic degeneration of the dopaminergic neurons in the substantia nigra (SN). The mechanisms proposed include oxidative (and nitrative) stress, inflammation, excitotoxicity, mitochondrial dysfunction, altered proteolysis and finally apoptotic induced cell death. Iron-mediated cellular destruction is mediated primarily via reactive oxygen or/and nitrogen species induced oxidative stress. Furthermore, these pathogenic mechanisms appear to be closely interlinked to the cascade of events leading to cellular death. There are conflicting reports about the stage during disease progression at which nigral iron change occurs in PD. Some have found that there are no changes in iron content SN in asymptomatic incidental Lewy body disease, suggesting it may represent a secondary event in the cascade of neuronal degeneration. In contrast, others have found an elevation of iron in SN in pre-clinical stages. These discrepancies may be attributed to the occurrence of different sub-groups of the disease. This concurs with the notion that PD represents a group of related diseases with a number of potential pathogenic pathways.     

Disruption of iron homeostasis and lung disease

As a result of a direct exchange with the external environment, the lungs are exposed to both iron and agents with a capacity to disrupt the homeostasis of this metal (e.g. particles). An increased availability of catalytically reactive iron can result from these exposures and, by generating an oxidative stress, this metal can contribute to tissue injury. By importing this Fe³⁺ into cells for storage in a chemically less reactive form, the lower respiratory tract demonstrates an ability to mitigate both the oxidative stress presented by iron and its potential for tissue injury. This means that detoxification is accomplished by chemical reduction to Fe²⁺ (e.g. by duodenal cytochrome b and other ferrireductases), iron import (e.g. by divalent metal transporter 1 and other transporters), and storage in ferritin. The metal can subsequently be exported from the cell (e.g. by ferroportin 1) in a less reactive state relative to that initially imported. Iron is then transported out of the lung via the mucociliary pathway or blood and lymphatic pathways to the reticuloendothelial system for long term storage. This coordinated handling of iron in the lung appears to be disrupted in several acute diseases on the lung including infections, acute respiratory distress syndrome, transfusion-related acute lung injury, and ischemia–reperfusion. Exposures to bleomycin, dusts and fibers, and paraquat similarly alter iron homeostasis in the lung to affect an oxidative stress. Finally, iron homeostasis is disrupted in numerous chronic lung diseases including pulmonary alveolar proteinosis, transplantation, cigarette smoking, and cystic fibrosis.     

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.     

The GDF‐5 mutant M1673 exerts robust anabolic and anti‐catabolic effects in chondrocytes

The growth and differentiation factor 5 (GDF‐5) is known to play a key role in cartilage morphogenesis and homeostasis, and a single‐nucleotide polymorphism in its promoter sequence was found to be associated with osteoarthritis (OA). In addition, GDF‐5 was shown to promote extracellular matrix (ECM) production in healthy chondrocytes, to stimulate chondrogenesis of mesenchymal stem cells (MSCs) and to protect against OA progression in vivo. Therefore, GDF‐5 appears to be a promising treatment for osteoarthritis. However, GDF‐5 also promotes osteogenesis and hypertrophy, limiting its therapeutic utility. To circumvent this, a GDF‐5 mutant with lower hypertrophic and osteogenic properties was engineered: M1673. The present study aimed to evaluate and compare the effects of GDF‐5 and M1673 on primary porcine and human OA chondrocytes. We found that both GDF‐5 and M1673 can robustly stimulate ECM accumulation, type II collagen and aggrecan expression in porcine and human OA chondrocytes in 3D culture. In addition, both molecules also down‐regulated MMP13 and ADAMTS5 expression. These results suggest that M1673 retained the anabolic and anti‐catabolic effects of GDF‐5 on chondrocytes and is an alternative to GDF‐5 for osteoarthritis.