4-hydroxy-2-nonenal and Osteoarthritis

Over the years, many theories have been proposed and examined to better explain the etiology and development of osteoarthritis (OA). The characteristics of joint destruction are one of the most important aspects in disease progression. Therefore, investigating different factors and signaling pathways involved in the alteration of extracellular matrix (ECM) turnover, and the subsequent catabolic damage to cartilage holds chief importance in understanding OA development. Among these factors, reactive oxygen species (ROS) have been at the forefront of the physiological and pathophysiological OA investigation.. In the last decades, research studies provided an enormous volume of data supporting the involvement of ROS in OA. Most interestingly, published data regarding the effect of exogenous antioxidant therapy in OA lack conclusive results from clinical trials to back up in vitro data. Accordingly, it is rational to suggest that there are other reactive species in OA that are not taken into account. Thus, our present review is focused on our current understanding of the involvement of lipid peroxidation-derived 4-hydroxynonenal (HNE) in OA.. Our findings, like those in the literature, illustrate the central role played by HNE in the regulation of a number of factors involved in joint homeostasis. HNE could thus be considered as an attractive therapeutic target in OA.

Topics: Aldehydes; Animals; Apoptosis; Chondrocytes; Humans; Lipid Peroxidation; Osteoarthritis; Oxidative Stress

Osteoarthritic chondrocytes show an over-activity of inflammatory catabolic mediators, and olive products have attracted attention because they were discovered to have some benefits on osteoarthritis patients. We investigated the mechanisms of action of olive leaf polyphenolic compounds in osteoarthritic chondrocytes (OACs) using a standardized leaf extract, ZeyEX, and its main phenolic component, oleuropein, also compared with anti-inflammatory drug ibuprofen. OACs, isolated from joint-cartilages of Grade 4 OA patients, were found to express COMP and MMP-9 throughout their culture period. ZeyEX, oleuropein, and ibuprofen increased cell viability at concentrations of 1-100 nM, did not change at 500 nM-50 μM, but inhibited at ≥100 μM. The adherence profile of OACs increased with 1 μM of ibuprofen or ZeyEX and 10 nM-1 μM oleuropein. Although the markers for oxidative and nitrosative stresses (ROS and 3-NT) generally inhibited by three agents, the inhibitory effect of ZeyEX on 3-NT emerged dramatically (1 nM-10 μM). Lipid-hydroperoxides and HNE-adducts were also inhibited by each agent, but AGE-adducts unchanged by oleuropein while reduced by ZeyEX and ibuprofen. Inflammatory biomarkers, IL-1β, IL-6, Casp-1/ICE, and TNF-α, were inhibited by three agents, however osteopontin and GM-CSF by only ZeyEX and ibuprofen. A decreased COMP, TLR4, and RAGE expression levels were observed by three agents, but only the effects of ZeyEX was concentration-dependent. In particular, ZeyEX and oleuropein improved COL2, inhibited p-JNK/JNK, and increased GPx. COX2 was only inhibited by ibuprofen. The results indicate that polyphenolic-olive compounds counteract redox-sensitive inflammatory aggressions in osteoarthritic chondrocytes that may stop the progression of pathology and allow regeneration.

Topics: Aged; Aldehydes; Biomarkers; Cartilage, Articular; Cell Adhesion; Cell Survival; Chondrocytes; Female; Glycation End Products, Advanced; Humans; Ibuprofen; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Lipid Peroxides; Male; Middle Aged; Olea; Osteoarthritis; Oxidation-Reduction; Phenol; Phosphorylation; Reactive Oxygen Species; Receptor for Advanced Glycation End Products; Toll-Like Receptor 4; Tyrosine

Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints.. Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated.. The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue.. Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Topics: Aged; Aged, 80 and over; Aldehydes; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Arthritis, Experimental; Cartilage, Articular; Chemokine CXCL1; Electron Transport Complex IV; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Injections, Intra-Articular; Interleukin-8; Knee Joint; Macrophages; Middle Aged; Mitochondria; Mitochondrial Proton-Translocating ATPases; NF-E2-Related Factor 2; Oligomycins; Osteoarthritis; Rats; Rats, Wistar; Reactive Oxygen Species; Synovial Membrane

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above-mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU.

Topics: Adult; Aged; Aged, 80 and over; Aldehydes; Alkaptonuria; Apoptosis; Cartilage; Chondrocytes; Enzyme Activation; Female; GTP-Binding Proteins; Humans; Joints; Male; Middle Aged; Osteoarthritis; Protein Glutamine gamma Glutamyltransferase 2; Spectrometry, X-Ray Emission; Staining and Labeling; Transglutaminases

Our study was designed to elucidate the precise molecular mechanisms by which sorbitol-modified hyaluronic acid (HA/sorbitol) exerts beneficial effects in osteoarthritis (OA).. Human OA chondrocytes were treated with increasing doses of HA/sorbitol ± anti-CD44 antibody or with sorbitol alone and thereafter with or without interleukin-1beta (IL-1β) or hydrogen peroxide (H2O2). Signal transduction pathways and parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated.. HA/sorbitol prevented IL-1β-induced oxidative stress, as measured by reactive oxygen species, p47-NADPH oxidase phosphorylation, 4-hydroxynonenal (HNE) production and HNE-metabolizing glutathione-S-transferase A4-4 expression. Moreover, HA/sorbitol stifled IL-1β-induced metalloproteinase-13, nitric oxide (NO) and prostaglandin E2 release as well as inducible NO synthase expression. Study of the apoptosis process revealed that this gel significantly attenuated cell death, caspase-3 activation and DNA fragmentation elicited by exposure to a cytotoxic H2O2 dose. Examination of signaling pathway components disclosed that HA/sorbitol prevented IL-1β-induced p38 mitogen-activated protein kinase and nuclear factor-kappa B activation, but not that of extracellular signal-regulated kinases 1 and 2. Interestingly, the antioxidant as well as the anti-inflammatory and anti-catabolic effects of HA/sorbitol were attributed to sorbitol and HA, respectively.. Altogether, our findings support a beneficial effect of HA/sorbitol in OA through the restoration of redox status and reduction of apoptosis, inflammation and catabolism involved in cartilage damage.

Topics: Aged; Aldehydes; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Cell Survival; Cells, Cultured; Chondrocytes; Dinoprostone; DNA Fragmentation; Glutathione Transferase; Humans; Hyaluronic Acid; Hydrogen Peroxide; Interleukin-1beta; Matrix Metalloproteinase 13; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis; Oxidative Stress; Reactive Oxygen Species; Sorbitol

Osteoarthritis (OA) is caused by the degradation of articular cartilage and affects approximately 80% of people over the age of 65. Matrix metalloproteinases (MMPs) belong to a group of zinc endopeptidases that degrade extracellular matrix (ECM) proteins in cartilage. MMP-13, also known as collagenase 3, cleaves type II collagen more rapidly than other MMPs and therefore is an important target for the treatment of OA. The lipid peroxidation product 4-hydroxy-2-(E)-nonenal (HNE), generated under oxidative stress, is known to play a crucial role in cartilage degradation; however, the mechanism is not yet fully understood. An approach has been developed to monitor HNE modification sites by incubating rhMMP-13 ± HNE in vitro followed by analysis of tryptic digests by UHPLC coupled to high resolution (HR) quadrupole-time-of-flight (QqTOF) tandem mass spectrometry (MS/MS). The analysis elucidated several covalently modified histidine and cysteine residues. The reaction was monitored using different HNE concentrations and incubation times. A targeted assay, using multiple-reaction monitoring (MRM), was then optimized to increase the sensitivity of detecting these modification sites in biological samples. HNE-related covalent modifications of MMP-13 were confirmed in enriched extracts from interleukin 1β-activated chondrocytes from OA patients using HR-MS/MS and MRM analysis.

Topics: Aldehydes; Amino Acid Sequence; Cells, Cultured; Chondrocytes; Chromatography, High Pressure Liquid; Humans; Immunoprecipitation; Interleukin-1beta; Matrix Metalloproteinase 13; Molecular Sequence Data; Osteoarthritis; Peptides; Recombinant Proteins; Tandem Mass Spectrometry

Nitric oxide (NO) and the lipid peroxidation (LPO) product 4-hydroxynonenal (HNE) are considered to be key mediators of cartilage destruction in osteoarthritis (OA). NO is also known to be an important intermediary in LPO initiation through peroxynitrite formation. The aim of the present study was to assess the ability of the inducible NO synthase (iNOS) inhibitor N-iminoethyl-L-lysine (L-NIL) to prevent HNE generation via NO suppression in human OA chondrocytes and cartilage explants. Human OA chondrocytes and cartilage explants were treated with L-NIL and thereafter with or without interleukin-1beta (IL-1β) or HNE at cytotoxic or non-cytotoxic concentrations. Parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. L-NIL stifled IL-1β-induced NO release, iNOS activity, nitrated proteins, and HNE generation in a dose-dependent manner. It also blocked IL-1β-induced inactivation of the HNE-metabolizing glutathione-s-transferase (GST). L-NIL restored both HNE and GSTA4-4 levels in OA cartilage explants. Interestingly, it also abolished IL-1β-evoked reactive oxygen species (ROS) generation and p47 NADPH oxidase activation. Furthermore, L-NIL significantly attenuated cell death and markers of apoptosis elicited by exposure to a cytotoxic dose of HNE as well as the release of prostaglandin E(2) and metalloproteinase-13 induced by a non-cytotoxic dose of HNE. Altogether, our findings support a beneficial effect of L-NIL in OA by (i) preventing the LPO process and ROS production via NO-dependent and/or independent mechanisms and (ii) attenuating HNE-induced cell death and different mediators of cartilage damage.

Topics: Aldehydes; Apoptosis; Cells, Cultured; Chondrocytes; Dinoprostone; Glutathione Transferase; Humans; Inflammation; Interleukin-1beta; Lipid Peroxidation; Lysine; Matrix Metalloproteinase 13; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis; Oxidative Stress; Reactive Oxygen Species

This study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).. For COX-2 and mPGES-1 studies, human osteoarthritic chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 muM HNE to the cultures at 2-hour intervals, up to 14 hours. For 5-LOX and FLAP studies, cells were treated with a single addition of 10 muM HNE for 24 hours, 48 hours, and 72 hours in the presence or absence of naproxen (a nonspecific COX-2 inhibitor) or antibody anti-transforming growth factor-beta 1 (TGF-beta1). The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by western blot, and those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-beta1 were determined with commercial kits. The levels of mPGES-1, FLAP and 5-LOX mRNA were measured by real-time RT-PCR. Transient transfection was performed to determine promoter activities of mPGES-1 and 5-LOX.. Single addition of 10 muM HNE to cultured chondrocytes induced PGE2 release as well as COX-2 and mPGES-1 expression at the protein and mRNA levels, with a plateau reached respectively at 8 and 16 hours of incubation, followed by a subsequent decline. However, repeated treatments with HNE prevented the decline of COX-2 and mPGES-1 expression that occurred with a single aldehyde addition. HNE induced mPGES-1 promoter activity, possibly through transcription factor Egr-1 activation. After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation. The addition of naproxen to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that HNE significantly induced TGF-beta1 production. The addition of anti-TGF-beta1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-beta1-dependent mechanism.. Our data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human osteoarthritic chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-beta1 are suggested to be involved in this regulation.

Topics: 5-Lipoxygenase-Activating Proteins; Aged; Aldehydes; Arachidonate 5-Lipoxygenase; Blotting, Western; Carrier Proteins; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Gene Expression; Gene Expression Regulation; Humans; Intramolecular Oxidoreductases; Leukotriene B4; Membrane Proteins; Microsomes; Osteoarthritis; Prostaglandin-E Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta1

Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects.. Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and α1β1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-κB-p65 levels were analyzed by Western blotting. The formation of α1β1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation.. Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, α1β1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-κB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited α1β1 integrin and Col II expression as well as ERK1/2 and NF-κB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of α1β1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug.. Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes.

Topics: Aged; Aldehydes; Blotting, Western; Carnosine; Cell Adhesion Molecules; Cell Separation; Cells, Cultured; Chondrocytes; Collagen Type II; Extracellular Matrix; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Profiling; Humans; Immunoprecipitation; Microscopy, Confocal; Osteoarthritis; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.. Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.. Our data showed that HNE at concentrations of up to 10 microM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 microM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.. Our study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.

Topics: Aged; Aldehydes; Apoptosis; Blotting, Western; Caspases; Cell Survival; Chondrocytes; Cysteine Proteinase Inhibitors; Glutathione Transferase; Humans; Microscopy, Fluorescence; Middle Aged; Osteoarthritis; Signal Transduction; Transfection

4-hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues and was recently identified as a potent catabolic factor in OA cartilage. In this study, we provide additional evidence that HNE acts as an inflammatory mediator by elucidating the signaling cascades targeted in OA chondrocytes leading to cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression. HNE induced COX-2 protein and mRNA levels with accompanying increases in prostaglandin E2 (PGE(2)) production. In contrast, HNE had no effect on basal iNOS expression or nitric oxide (NO) release. However, HNE strongly inhibited IL-1beta-induced iNOS or NO production. Transient transfection experiments revealed that the ATF/CRE site (-58/-53) is essential for HNE-induced COX-2 promoter activation and indeed HNE induced ATF-2 and CREB-1 phosphorylation as well as ATF/CRE binding activity. Overexpression of p38 MAPK enhanced the HNE-induced ATF/CRE luciferase reporter plasmid activation, COX-2 synthesis and promoter activity. HNE abrogated IL-1beta-induced iNOS expression and promoter activity mainly through NF-kappaB site (-5,817/-5,808) possibly via suppression of IKKalpha-induced IkappaBalpha phosphorylation and NF-kappaB/p65 nuclear translocation. Upon examination of upstream signaling components, we found that IKKalpha was inactivated through HNE/IKKalpha adduct formation. Taken together, these findings illustrate the central role played by HNE in the regulation of COX-2 and iNOS in OA. The aldehyde induced selectively COX-2 expression via ATF/CRE activation and inhibited iNOS via IKKalpha inactivation.

Topics: Activating Transcription Factor 2; Aldehydes; Blotting, Western; Cartilage; Chondrocytes; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Immunoprecipitation; Membrane Proteins; Middle Aged; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcriptional Activation

The lipid peroxidation product 4-hydroxynonenal (HNE) is prominently produced in osteoarthritic (OA) synovial cells, but its specific contribution to cartilage destruction is not understood. This study was designed to test whether HNE signaling and binding are involved in OA cartilage degradation through type II collagen (CII) and matrix metalloproteinase 13 (MMP-13) modulation.. HNE levels in synovial fluid and in isolated OA chondrocytes treated with free radical donors were determined by enzyme-linked immunosorbent assay. The formation of the HNE/CII adducts was measured in cartilage explants by immunoprecipitation. Levels of CII and MMP-13 messenger RNA and protein were determined by reverse transcription-polymerase chain reaction, Western blotting, and by the use of commercial kits.. Levels of HNE/protein adducts were higher in OA synovial fluid compared with normal synovial fluid and were higher in OA chondrocytes treated with free radical donors compared with untreated cells. In cartilage explants, HNE induced CII cleavage, as established by the generation of neoepitopes. The level of HNE/CII adducts was increased in OA cartilage explants incubated with free radical donors. Modification of CII by HNE accelerated its degradation by active MMP-13. In isolated OA chondrocytes, HNE inhibited the expression of CII and tissue inhibitor of metalloproteinases 1 and induced MMP-13 mainly through activation of p38 MAPK. In vitro, HNE binding to MMP-13 activated this enzyme at a molar ratio of 1:100 (MMP-13 to HNE).. The increased level of HNE in OA cartilage and the ability of HNE to induce transcriptional and posttranslational modifications of CII and MMP-13 suggest that this aldehyde could play a role in OA.

Topics: Aged; Aldehydes; Cartilage, Articular; Collagen Type II; Collagenases; Humans; Lipid Peroxidation; Matrix Metalloproteinase 13; Middle Aged; Osteoarthritis

4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H2O2. Investigating osteoblast markers, we found that HNE increased osteocalcin and type I collagen synthesis but inhibited alkaline phosphatase activity. We next examined the effects of HNE on the signaling pathways controlling cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) expression in view of their putative role in OA pathophysiology. HNE dose-dependently decreased basal and tumour necrosis factor-alpha (TNF-alpha)-induced IL-6 expression while inducing COX-2 expression and prostaglandin E2 (PGE2) release. In a similar pattern, HNE induces changes in osteoblast markers as well as PGE2 and IL-6 release in normal osteoblasts. Upon examination of signaling pathways involved in PGE2 and IL-6 production, we found that HNE-induced PGE2 release was abrogated by SB202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Overexpression of p38 MAPK enhanced HNE-induced PGE2 release. In this connection, HNE markedly increased the phosphorylation of p38 MAPK, JNK2, and transcription factors (CREB-1, ATF-2) with a concomitant increase in the DNA-binding activity of CRE/ATF. Transfection experiments with a human COX-2 promoter construct revealed that the CRE element (-58/-53 bp) was essential for HNE-induced COX-2 promoter activity. However, HNE inhibited the phosphorylation of IkappaBalpha and subsequently the DNA-binding activity of nuclear factor-kappaB. Overexpression of IKKalpha increased TNF-alpha-induced IL-6 production. This induction was inhibited when TNF-alpha was combined with HNE. These findings suggest that HNE may exert multiple effects on human OA osteoblasts by selective activation of signal transduction pathways and alteration of osteoblastic phenotype expression and pro-inflammatory mediator production.

Topics: Aldehydes; Blotting, Western; Cell Line; Cyclooxygenase 2; Dinoprostone; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Interleukin-6; Lipid Peroxidation; Osteoarthritis; Osteoblasts; Oxidants; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Necrosis Factor-alpha

To investigate the role of oxidative functions in human osteoarthritic (OA) chondrocytes and to investigate the presence of in vivo molecular markers of lipoxidation in OA cartilage.. An in vitro model of cartilage collagen degradation was used. Lipid peroxidation activity and overall oxidative function in OA chondrocytes were monitored by cis-parinaric acid and dichlorofluorescein assays, respectively. In vivo molecular markers of lipoxidation in normal and OA cartilage were studied using immunohistochemistry to detect the presence of malondialdehyde and hydroxynonenal adducts.. Human OA chondrocytes showed a robust amount of 3H-proline-labeled collagen degradation upon stimulation with lipopolysaccharide and calcium ionophore A21387, as compared with that in untreated OA chondrocytes. Primary OA chondrocytes showed both spontaneous and inducible levels of lipid peroxidation activity. However, lipid peroxidation activity was already maximally elevated in more than 50% of the OA chondrocyte samples. Overall, spontaneous and inducible oxidative activities were observed in all OA samples. Immunohistochemical analysis of control OA tissue sections that were not treated with monoclonal antibody showed little immunoreactivity. OA cartilage sections treated with monoclonal antibodies showed specific immunoreactivity on the cartilage surface, at sites of OA lesions, at the pericellular matrix, and at intra- and intercellular matrices. Normal cartilage sections showed faint surface reactivity.. Our observations suggest that human OA chondrocytes demonstrate spontaneous and inducible cell-associated lipoxidative and nonlipoxidative activity. Lipoxidative activity appears to be enhanced in OA chondrocytes. The presence of molecular markers of in vivo lipid peroxidation was demonstrated in OA cartilage, suggesting its role in the pathogenesis of the disease.

Topics: Adult; Aged; Aldehydes; Antibodies, Monoclonal; Biomarkers; Calcimycin; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagen; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Fluoresceins; Humans; Hydrogen Peroxide; Immunohistochemistry; Ionophores; Joints; Lipid Peroxidation; Lipopolysaccharides; Malondialdehyde; Middle Aged; Osteoarthritis

To evaluate the degree of lipid peroxidation of synoviocytes from patients with rheumatoid arthritis (RA), osteoarthitis (OA), and controls and to look at the production of nitric oxide (NO) and its involvement in this process.. Human synoviocytes were isolated from synovial tissues from patients with RA, OA, and from healthy controls. Cells were maintained in culture for up to 3 culture passages. Lipid peroxidation, verified by the production of malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE), was determined by colorimetric assay. NO was evaluated by estimating the stable NO metabolite nitrite by the Griess method in the supernatants of unstimulated and interleukin (IL)-1beta and tumor necrosis factor (TNF)-a stimulated cells.. Increased levels of lipid peroxidation were observed for OA-derived synoviocytes compared to RA and controls. The cells in each experimental group produced low amounts of NO both in basal and in stimulated conditions.. In OA, synovial cells underwent a lipid peroxidation process that did not occur in synoviocytes from RA or controls even in the absence of a detectable production of the reactive nitrogen intermediate NO. We can postulate that this peroxidation process might be due to the action of NO secreted by chondrocytes that are known to produce higher levels of this radical in OA compared to RA.

Topics: Adult; Aged; Aged, 80 and over; Aldehydes; Arthritis, Rheumatoid; Cells, Cultured; Humans; In Vitro Techniques; Lipid Peroxidation; Malondialdehyde; Middle Aged; Nitric Oxide; Osteoarthritis; Synovial Membrane

(E)-4-Hydroxy-2-nonenal (HNE), a cytotoxic propagation product of lipid peroxidation, is present in the synovial fluid (0.54 (0.19) mumol/l; mean (SE), n = 9) and plasma (0.34 (0.09) mumol/l, n = 9) of patients with rheumatoid arthritis. This compound was also found in the synovial fluid (0.24 (0.19) mumol/l, n = 9) and plasma (0.09 (0.03) mumol/l, n = 9) of patients with osteoarthritis. The concentration of HNE in the plasma of patients with rheumatoid arthritis was significantly greater than in patients with osteoarthritis.

Topics: Aldehydes; Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Lipid Peroxidation; Osteoarthritis; Synovial Fluid