Development of New Resolvin D1 Analogues for Osteoarthritis Therapy: Acellular and Computational Approaches to Study Their Antioxidant Activities.

In osteoarthritis (OA), oxidative stress plays a crucial role in maintaining and sustaining cartilage degradation. Current OA management requires a combination of pharmaceutical and non-pharmacological strategies, including intraarticular injections of hyaluronic acid (HA). However, several lines of evidence reported that HA oxidation by reactive oxygen species (ROS) is linked with HA cleavage and fragmentation, resulting in reduced HA viscosity. Resolvin D1 (RvD1) is a lipid mediator that is biosynthesized from omega-3 polyunsaturated fatty acids and is a good candidate with the potential to regulate a panoply of biological processes, including tissue repair, inflammation, oxidative stress, and cell death in OA. Herein, newly designed and synthesized imidazole-derived RvD1 analogues were introduced to compare their potential antioxidant properties with commercially available RvD1. Their antioxidant capacities were investigated by several in vitro chemical assays including oxygen radical absorbance capacity, 2,2-diphenyl-1-picrylhydrazyl radical scavenging, ferric ion reducing antioxidant power, hydroxyl radical scavenging, and HA fragmentation assay. All results proved that imidazole-derived RvD1 analogues showed excellent antioxidant performance compared to RvD1 due to their structural modifications. Interestingly, they scavenged the formed reactive oxygen species (ROS) and protected HA from degradation, as verified by agarose gel electrophoresis and gel permission chromatography. A computational study using Gaussian 09 with DFT calculations and a B3LYP/6-31 G (d, p) basis set was also employed to study the relationship between the antioxidant properties and chemical structures as well as calculation of the molecular structures, frontier orbital energy, molecular electrostatic potential, and bond length. The results showed that the antioxidant activity of our analogues was higher than that of RvD1. In conclusion, the findings suggest that imidazole-derived RvD1 analogues can be good candidates as antioxidant molecules for the treatment of oxidative stress-related diseases like OA. Therefore, they can prolong the longevity of HA in the knee and thus may improve the mobility of the articulation.

Role of epigenetics and the transcription factor Sp1 in the expression of the D prostanoid receptor 1 in human cartilage.

D prostanoid receptor 1 (DP1), a prostaglandin D2 receptor, plays a central role in the modulation of inflammation and cartilage metabolism. We have previously shown that activation of DP1 signaling downregulated catabolic responses in cultured chondrocytes and was protective in mouse osteoarthritis (OA). However, the mechanisms underlying its transcriptional regulation in cartilage remained poorly understood. In the present study, we aimed to characterize the human DP1 promoter and the role of DNA methylation in DP1 expression in chondrocytes. In addition, we analyzed the expression level and methylation status of the DP1 gene promoter in normal and OA cartilage. Deletion and site-directed mutagenesis analyses identified a minimal promoter region (-250/-120) containing three binding sites for specificity protein 1 (Sp1). Binding of Sp1 to the DP1 promoter was confirmed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Treatment with the Sp1 inhibitor mithramycin A reduced DP1 promoter activity and DP1 mRNA expression. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine upregulated DP1 expression, and in vitro methylation reduced the DP1 promoter activity. Neither the methylation status of the DP1 promoter nor the DP1 expression level were different between normal and OA cartilage. In conclusion, our results suggest that the transcription factor Sp1 and DNA methylation are important determinants of DP1 transcription regulation. They also suggest that the methylation status and expression level of DP1 are not altered in OA cartilage. These findings will improve our understanding of the regulatory mechanisms of DP1 transcription and may facilitate the development of intervention strategies involving DP1.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

OBJECTIVE AND DESIGN: 15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. METHODS: Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. RESULTS: Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. CONCLUSION: The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

Impact of Degree of Ionization and PEGylation on the Stability of Nanoparticles of Chitosan Derivatives at Physiological Conditions.

Nowadays, the therapeutic efficiency of small interfering RNAs (siRNA) is still limited by the efficiency of gene therapy vectors capable of carrying them inside the target cells. In this study, siRNA nanocarriers based on low molecular weight chitosan grafted with increasing proportions (5 to 55%) of diisopropylethylamine (DIPEA) groups were developed, which allowed precise control of the degree of ionization of the polycations at pH 7.4. This approach made obtaining siRNA nanocarriers with small sizes (100-200 nm), positive surface charge and enhanced colloidal stability (up to 24 h) at physiological conditions of pH (7.4) and ionic strength (150 mmol L-1) possible. Moreover, the PEGylation improved the stability of the nanoparticles, which maintained their colloidal stability and nanometric sizes even in an albumin-containing medium. The chitosan-derivatives displayed non-cytotoxic effects in both fibroblasts (NIH/3T3) and macrophages (RAW 264.7) at high N/P ratios and polymer concentrations (up to 0.5 g L-1). Confocal microscopy showed a successful uptake of nanocarriers by RAW 264.7 macrophages and a promising ability to silence green fluorescent protein (GFP) in HeLa cells. These results were confirmed by a high level of tumor necrosis factor-α (TNFα) knockdown (higher than 60%) in LPS-stimulated macrophages treated with the siRNA-loaded nanoparticles even in the FBS-containing medium, findings that reveal a good correlation between the degree of ionization of the polycations and the physicochemical properties of nanocarriers. Overall, this study provides an approach to enhance siRNA condensation by chitosan-based carriers and highlights the potential of these nanocarriers for in vivo studies.

Chitosan-Based Nanogels: Synthesis and Toxicity Profile for Drug Delivery to Articular Joints.

One important challenge in treating avascular-degraded cartilage is the development of new drugs for both pain management and joint preservation. Considerable efforts have been invested in developing nanosystems using biomaterials, such as chitosan, a widely used natural polymer exhibiting numerous advantages, i.e., non-toxic, biocompatible and biodegradable. However, even if chitosan is generally recognized as safe, the safety and biocompatibility of such nanomaterials must be addressed because of potential for greater interactions between nanomaterials and biological systems. Here, we developed chitosan-based nanogels as drug-delivery platforms and established an initial biological risk assessment for osteocartilaginous applications. We investigated the influence of synthesis parameters on the physicochemical characteristics of the resulting nanogels and their potential impact on the biocompatibility on all types of human osteocartilaginous cells. Monodisperse nanogels were synthesized with sizes ranging from 268 to 382 nm according to the acidic solution used (i.e., either citric or acetic acid) with overall positive charge surface. Our results demonstrated that purified chitosan-based nanogels neither affected cell proliferation nor induced nitric oxide production in vitro. However, nanogels were moderately genotoxic in a dose-dependent manner but did not significantly induce acute embryotoxicity in zebrafish embryos, up to 100 µg∙mL-1. These encouraging results hold great promise for the intra-articular delivery of drugs or diagnostic agents for joint pathologies.

Lymphocyte transformation test reveals low prevalence of true metal hypersensitivity among pre-operative total knee arthroplasty patients.

PURPOSE: The aims of this study were to determine the prevalence of metal hypersensitivity, and identify pre-operative factors which could predict susceptibility to hypersensitivity reactions among patients scheduled for primary total knee arthroplasty (TKA). The present study used a testing method consistent with the recognised biological response to metals. METHODS: A prospective cross-sectional analysis of 220 patients was conducted. All patients received a testing protocol using lymphocyte transformation test to evaluate reactivity to possible contents of orthopaedic implants. Test response is interpreted as stimulation index (SI) values. A comprehensive questionnaire was used to evaluate prior exposure. Patients were categorised according to SI values and the odds ratios (OR) were calculated as comparative effect measure for each predetermined prior exposure factor. RESULTS: The prevalence of metal sensitivity response was 28% (n = 61) among patients with susceptibility to at least one agent (SI = 2 to 4.9), and 3.2% (n = 7) among patients with true hypersensitivity (SI ≥ 5). The population-weighted prevalence, adjusted for sampling weights of symptomatic knee osteoarthritis, was SI ≥ 5 = 4.7% (95% CI 0.4-11.8%) and SI ≥ 2 = 35.2% (95% CI 24.8-48.6%). Stimulation index levels of response to materials were markedly varied with the highest being aluminium. Female sex, smoking history, cutaneous reaction to jewellery, occupational exposure, and dental procedures were among factors shown to increase the odds of having higher reactivity response to tested metals. Nevertheless, patients with well-functioning prior contralateral TKA did not appear at greater risk of having either sensitivity or susceptibility with odds ratio (OR) = 0.2 (95% CI 0.01-3.2), p: NS and OR = 0.6 (95% CI 0.3-1.2), p: NS, respectively. Prior positive patch test was neither predictor of susceptibility to hypersensitivity OR = 1.2 (95% CI 0.6-2.6) p: NS nor predictor of true hypersensitivity OR = 0.7 (95% CI 0.08-6.1), p: NS. CONCLUSION: Among patients scheduled for primary TKA with no prior clinical features of metal allergy the prevalence of true hypersensitivity to at least one metal is just over 3%. Patients are likely to encounter a material to which they have pre-existing susceptibility to hypersensitivity. With certain prior exposure factors, there was increased susceptibility to metal hypersensitivity reaction evoking an acquired condition. LEVEL OF EVIDENCE: Level II, prospective cross-sectional study.

The pathophysiology of immunoporosis: innovative therapeutic targets.

BACKGROUND: The physiological balance between bone resorption and bone formation is now known to be mediated by a cascade of events parallel to the classic osteoblast-osteoclast interaction. Thus, osteoimmunology now encompasses the role played by other cell types, such as cytokines, lymphocytes and chemokines, in immunological responses and how they help modulate bone metabolism. All these factors have an impact on the RANK/RANKL/OPG pathway, which is the major pathway for the maturation and resorption activity of osteoclast precursor cells, responsible for osteoporosis development. Recently, immunoporosis has emerged as a new research area in osteoimmunology dedicated to the immune system's role in osteoporosis. METHODS: The first part of this review presents theoretical concepts on the factors involved in the skeletal system and osteoimmunology. Secondly, existing treatments and novel therapeutic approaches to treat osteoporosis are summarized. These were selected from to the most recent studies published on PubMed containing the term osteoporosis. All data relate to the results of in vitro and in vivo studies on the osteoimmunological system of humans, mice and rats. FINDINGS: Treatments for osteoporosis can be classified into two categories. They either target osteoclastogenesis inhibition (denosumab, bisphosphonates), or they aim to restore the number and function of osteoblasts (romozumab, abaloparatide). Even novel therapies, such as resolvins, gene therapy, and mesenchymal stem cell transplantation, fall within this classification system. CONCLUSION: This review presents alternative pathways in the pathophysiology of osteoporosis, along with some recent therapeutic breakthroughs to restore bone homeostasis.

Identification of 4-hydroxynonenal-modified proteins in human osteoarthritic chondrocytes.

The α,ß-unsaturated aldehyde 4-hydroxynonenal (HNE) is formed through lipid peroxidation during oxidative stress. As a highly reactive electrophile, it is able to form adducts with various biomolecules, including proteins. These protein modifications could modulate many signaling pathways, as well as cell differentiation and proliferation, and thus could be highly important in the context of the extracellular matrix and degradation of articular cartilage. This study specifically investigated the role of HNE as a bioactive molecule in chondrocytes of osteoarthritis (OA) patients. Chondrocyte extracts of OA and non-OA patients were analyzed for HNE binding using Western blot and bottom-up LC-MS/MS analyses. HNE-modified histones, H2A and H2B, and histone deacetylase were identified using anti-HNE antibodies. Furthermore, peptide sequencing and database searching revealed 95 distinct HNE-modified proteins and their exact modification sites, with 88 protein adducts being unique to OA chondrocytes. HNE-proteins of specific interest included histone H2A, H2B and H4, collagen alpha-3(VI) chain, eukaryotic initiation factor 4A-I, and nucleolar RNA helicase 2. Comparing their MS/MS spectra to those of HNE-modified standard peptides further validated the six HNE-proteins. SIGNIFICANCE: HNE binding to proteins has been shown to result in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in OA development. Considering the increased levels of HNE in OA cartilage, this reactive aldehyde could play a role in OA. This work represents a clinically-relevant in vivo study to demonstrate the pathophysiological role of HNE in human OA. Since HNE binding can alter protein conformation and function, it remains highly relevant to study the effects of this modification in OA.

L-PGDS deficiency accelerated the development of naturally occurring age-related osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder among the elderly. It is characterized by progressive cartilage degradation, synovial inflammation, subchondral bone remodeling and pain. Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, which has been implicated in the regulation of inflammation and cartilage biology. This study aimed to evaluate the effect of L-PGDS deficiency on the development of naturally occurring age-related OA in mice. OA-like structural changes were assessed by histology, immunohistochemistry, and micro-computed tomography. Pain related behaviours were assessed using the von Frey and the open-field assays. L-PGDS deletion promoted cartilage degradation during aging, which was associated with enhanced expression of extracellular matrix degrading enzymes, matrix metalloprotease 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and their breakdown products, C1,2C, VDIPEN and NITEG. Moreover, L-PGDS deletion enhanced subchondral bone changes, but had no effect on its angiogenesis. Additionally, L-PGDS deletion increased mechanical sensitivity and reduced spontaneous locomotor activity. Finally, we showed that the expression of L-PGDS was elevated in aged mice. Together, these findings indicate an important role for L-PGDS in naturally occurring age-related OA. They also suggest that L-PGDS may constitute a new efficient therapeutic target in OA.

Evidence Supporting the Safety of Pegylated Diethylaminoethyl-Chitosan Polymer as a Nanovector for Gene Therapy Applications.

PURPOSE: Diethylaminoethyl-chitosan (DEAE-CH) is a derivative with excellent potential as a delivery vector for gene therapy applications. The aim of this study is to evaluate its toxicological profile for potential future clinical applications. METHODS: An endotoxin-free chitosan (CH) modified with DEAE, folic acid (FA) and polyethylene glycol (PEG) was used to complex small interfering RNA (siRNA) and form nanoparticles (DEAE12-CH-PEG-FA2/siRNA). Based on the guidelines from the International Organization for Standardization (ISO), the American Society for Testing and Materials (ASTM), and the Nanotechnology Characterization Laboratory (NCL), we evaluated the effects of the interaction between these nanoparticles and blood components. In vitro screening assays such as hemolysis, hemagglutination, complement activation, platelet aggregation, coagulation times, cytokine production, and reactive species, such as nitric oxide (NO) and reactive oxygen species (ROS), were performed on erythrocytes, plasma, platelets, peripheral blood mononuclear cells (PBMC) and Raw 264.7 macrophages. Moreover, MTS and LDH assays on Raw 264.7 macrophages, PBMC and MG-63 cells were performed. RESULTS: Our results show that a targeted theoretical plasma concentration (TPC) of DEAE12-CH-PEG-FA2/siRNA nanoparticles falls within the guidelines' thresholds: <1% hemolysis, 2.9% platelet aggregation, no complement activation, and no effect on coagulation times. ROS and NO production levels were comparable to controls. Cytokine secretion (TNF-α, IL-6, IL-4, and IL-10) was not affected by nanoparticles except for IL-1ß and IL-8. Nanoparticles showed a slight agglutination. Cell viability was >70% for TPC in all cell types, although LDH levels were statistically significant in Raw 264.7 macrophages and PBMC after 24 and 48 h of incubation. CONCLUSION: These DEAE12-CH-PEG-FA2/siRNA nanoparticles fulfill the existing ISO, ASTM and NCL guidelines' threshold criteria, and their low toxicity and blood biocompatibility warrant further investigation for potential clinical applications.

Role of Lipocalin-Type Prostaglandin D Synthase in Experimental Osteoarthritis.

OBJECTIVE: Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the formation of prostaglandin D2 (PGD2 ), which has important roles in inflammation and cartilage metabolism. We undertook this study to investigate the role of L-PGDS in the pathogenesis of osteoarthritis (OA) using an experimental mouse model. METHODS: Experimental OA was induced in wild-type (WT) and L-PGDS-deficient (L-PGDS-/- ) mice (n = 10 per genotype) by destabilization of the medial meniscus (DMM). Cartilage degradation was evaluated by histology. The expression of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 was assessed by immunohistochemistry. Bone changes were determined by micro-computed tomography. Cartilage explants from L-PGDS-/- and WT mice (n = 6 per genotype) were treated with interleukin-1α (IL-1α) ex vivo in order to evaluate proteoglycan degradation. Moreover, the effect of intraarticular injection of a recombinant adeno-associated virus type 2/5 (rAAV2/5) encoding L-PGDS on OA progression was evaluated in WT mice (n = 9 per group). RESULTS: Compared to WT mice, L-PGDS-/- mice had exacerbated cartilage degradation and enhanced expression of MMP-13 and ADAMTS-5 (P < 0.05). Furthermore, L-PGDS-/- mice displayed increased synovitis and subchondral bone changes (P < 0.05). Cartilage explants from L-PGDS-/- mice showed enhanced proteoglycan degradation following treatment with IL-1α (P < 0.05). Intraarticular injection of rAAV2/5 encoding L-PGDS attenuated the severity of DMM-induced OA-like changes in WT mice (P < 0.05). The L-PGDS level was increased in OA tissues of WT mice (P < 0.05). CONCLUSION: Collectively, these findings suggest a protective role of L-PGDS in OA, and therefore enhancing levels of L-PGDS may constitute a promising therapeutic strategy.

In vitro and in vivo assessment of the proresolutive and antiresorptive actions of resolvin D1: relevance to arthritis.

BACKGROUND: Resolvin D1 (RvD1), an important member of resolvins, exerts a wide spectrum of biological effects, including resolution of inflammation, tissue repair, and preservation of cell viability. The aim of the present study is to investigate the anti-arthritic potential and clarify the bone protective actions of RvD1 in vitro and in vivo. METHODS: RAW264.7 cells were treated with 50 ng/ml LPS for 72 h in the presence or absence of RvD1 (0-500 nM). Primary human monocytes were treated with M-CSF + RANKL for 14 days ± RvD1 (0-500 nM) with or without siRNA against RvD1 receptor FPR2. Expressions of inflammatory mediators, degrading enzymes, osteoclasts (OC) formation, and bone resorption were analyzed. The therapeutic effect of RvD1 (0-1000 ng) was carried out in murine collagen antibody-induced arthritis. Arthritis scoring, joint histology, and inflammatory and bone turnover markers were measured. RESULTS: RvD1 is not toxic and inhibits OC differentiation and activation. It decreases bone resorption, as assessed by the inhibition of TRAP and cathepsin K expression, hydroxyapatite matrix resorption, and bone loss. In addition, RvD1 reduces TNF-α, IL-1ß, IFN-γ, PGE2, and RANK and concurrently enhances IL-10 in OC. Moreover, in arthritic mice, RvD1 alleviates clinical score, paw inflammation, and bone and joint destructions. Besides, RvD1 reduces inflammatory mediators and markedly decreases serum markers of bone and cartilage turnover. CONCLUSION: Our results provide additional evidence that RvD1 plays a key role in preventing bone resorption and other pathophysiological changes associated with arthritis. The study highlights the clinical relevance of RvD1 as a potential compound for the treatment of inflammatory arthritis and related bone disorders.

Diethylaminoethyl- chitosan as an efficient carrier for siRNA delivery: Improving the condensation process and the nanoparticles properties.

Chitosan has been indicated as a promising carrier for the preparation of small interfering RNA (siRNA) delivery systems due to its remarkable properties. However, its weak interactions with siRNA molecules makes the condensation of siRNA molecules into nanoparticles difficult. In this work, a non-viral gene delivery system based on diethylaminoethyl chitosan (DEAE-CH) derivatives of varied Mw (25-230 kDa) having a low degree of substitution of 15% was investigated. The presence of secondary and tertiary amino groups strengthened the interaction of siRNA and DEAE-CH derivatives of higher Mw (130 kDa to 230 kDa) and provided the preparation of spherical nanoparticles at low charge ratios (N/P 2 to 3) with low polydispersities (0.15 to 0.2) in physiological ionic strength. Nanoparticles prepared with all derivatives exhibited remarkable silencing efficiencies (80% to 90%) on different cell lines (HeLa, MG-63, OV-3) by adjusting the charge ratios. A selected PEG-folic acid labeled derivative (FA-PEG-DEAE15-CH230) was synthesized and its nanoparticles completely inhibited the mRNA expression level of TNF-α in RAW 264.7 macrophages. The study demonstrates that the insertion of DEAE groups provides improved physical properties to chitosan-siRNA nanoparticles and holds potential for in vivo applications.

In vivo therapeutic efficacy of TNFα silencing by folate-PEG-chitosan-DEAE/siRNA nanoparticles in arthritic mice.

BACKGROUND: Tumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine, has been shown to play a role in the pathophysiology of rheumatoid arthritis. Silencing TNFα expression with small interfering RNA (siRNA) is a promising approach to treatment of the condition. METHODS: Towards this end, our team has developed a modified chitosan (CH) nanocarrier, deploying folic acid, diethylethylamine (DEAE) and polyethylene glycol (PEG) (folate-PEG-CH-DEAE15). The gene carrier protects siRNA against nuclease destruction, its ligands facilitate siRNA uptake via cell surface receptors, and it provides improved solubility at neutral pH with transport of its load into target cells. In the present study, nanoparticles were prepared with siRNA-TNFα, DEAE, and folic acid-CH derivative. Nanoparticle size and zeta potential were verified by dynamic light scattering. Their TNFα-knockdown effects were tested in a murine collagen antibody-induced arthritis model. TNFα expression was examined along with measurements of various cartilage and bone turnover markers by performing histology and microcomputed tomography analysis. RESULTS: We demonstrated that folate-PEG-CH-DEAE15/siRNA nanoparticles did not alter cell viability, and significantly decreased inflammation, as demonstrated by improved clinical scores and lower TNFα protein concentrations in target tissues. This siRNA nanocarrier also decreased articular cartilage destruction and bone loss. CONCLUSION: The results indicate that folate-PEG-CH-DEAE15 nanoparticles are a safe and effective platform for nonviral gene delivery of siRNA, and their potential clinical applications warrant further investigation.

Exacerbation of Aging-Associated and Instability-Induced Murine Osteoarthritis With Deletion of D Prostanoid Receptor 1, a Prostaglandin D2 Receptor.

OBJECTIVE: D prostanoid receptor 1 (DP1), a receptor for prostaglandin D2 , plays important roles in inflammation and cartilage metabolism. However, its role in the pathogenesis of osteoarthritis (OA) remains unknown. This study was undertaken to explore the roles of DP1 in the development of OA in murine models and to evaluate the efficacy of a DP1 selective agonist in the treatment of OA. METHODS: The development of aging-associated OA and destabilization of the medial meniscus (DMM)-induced OA was compared between DP1-deficient (DP1-/- ) and wild-type (WT) mice. The progression of OA was assessed by histology, immunohistochemistry, and micro-computed tomography. Cartilage explants from DP1-/- and WT mice were treated with interleukin-1α (IL-1α) ex vivo, to evaluate proteoglycan degradation. The effect of intraperitoneal administration of the DP1 selective agonist BW245C on OA progression was evaluated in WT mice. RESULTS: Compared to WT mice, DP1-/- mice had exacerbated cartilage degradation in both models of OA, and this was associated with increased expression of matrix metalloproteinase 13 and ADAMTS-5. In addition, DP1-/- mice demonstrated enhanced subchondral bone changes. Cartilage explants from DP1-/- mice showed enhanced proteoglycan degradation following treatment with IL-1α. Intraperitoneal injection of BW245C attenuated the severity of DMM-induced cartilage degradation and bony changes in WT mice. CONCLUSION: These findings indicate a critical role for DP1 signaling in OA pathogenesis. Modulation of the functions of DP1 may constitute a potential therapeutic target for the development of novel OA treatments.

An overview of the role of lipid peroxidation-derived 4-hydroxynonenal in osteoarthritis.

BACKGROUND: 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. FINDINGS: 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. CONCLUSION: 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.

Elucidating the Role of Protandim and 6-Gingerol in Protection Against Osteoarthritis.

Protandim and 6-gingerol, two potent nutraceuticals, have been shown to decrease free radicals production through enhancing endogenous antioxidant enzymes. In this study, we evaluated the effects of these products on the expression of different factors involved in osteoarthritis (OA) process. Human OA chondrocytes were treated with 1 ng/ml IL-1ß in the presence or absence of protandim (0-10 µg/ml) or 6-gingerol (0-10 µM). OA was induced surgically in mice by destabilization of the medial meniscus (DMM). The animals were treated weekly with an intraarticular injection of 10 µl of vehicle or protandim (10 µg/ml) for 8 weeks. Sham-operated mice served as controls. In vitro, we demonstrated that protandim and 6-gingerol preserve cell viability and mitochondrial metabolism and prevented 4-hydroxynonenal (HNE)-induced cell mortality. They activated Nrf2 transcription factor, abolished IL-1ß-induced NO, PGE2 , MMP-13, and HNE production as well as IL-ß-induced GSTA4-4 down-regulation. Nrf2 overexpression reduced IL-1ß-induced HNE and MMP-13 as well as IL-1ß-induced GSTA4-4 down-regulation. Nrf2 knockdown following siRNA transfection abolished protandim protection against oxidative stress and catabolism. The activation of MAPK and NF-κB by IL-1ß was not affected by 6-gingerol. In vivo, we observed that Nrf2 and GSTA4-4 expression was significantly lower in OA cartilage from humans and mice compared to normal controls. Interestingly, protandim administration reduced OA score in DMM mice. Altogether, our data indicate that protandim and 6-gingerol are essential in preserving cartilage and abolishing a number of factors known to be involved in OA pathogenesis. J. Cell. Biochem. 118: 1003-1013, 2017. © 2016 Wiley Periodicals, Inc.

The role of resolvin D1 in the regulation of inflammatory and catabolic mediators in osteoarthritis.

OBJECTIVE AND DESIGN: Resolvin D1 (RvD1), an omega-3 fatty acid derivative, has shown remarkable properties in resolving inflammation, promoting tissue repair and preserving tissue integrity. In this study, we investigated RvD1 effects on major processes involved in osteoarthritis (OA) pathophysiology. MATERIALS AND METHODS: Human OA chondrocytes were treated with either 1 ng/ml interleukin-1ß (IL-1ß) or 20 µM 4-hydroxynonenal (HNE), then treated or not with increased concentrations of RvD1 (0-10 µM). RvD1 levels were measured by enzyme immunoassay in synovial fluids from experimental dog model of OA and sham operated dogs obtained from our previous study. Cell viability was evaluated by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide assay. Parameters related to inflammation, catabolism and apoptosis were determined by enzyme-linked immunosorbent assay, Western blotting, and quantitative polymerase chain reaction. Glutathione (GSH) was assessed by commercial kit. The activation of mitogen-activated protein kinases and nuclear factor-kappaB (NF-κB) pathways was evaluated by Western blot. RESULTS: We showed that RvD1 levels were higher in synovial fluids from OA joint compared to controls. In OA human chondrocytes, we demonstrated that RvD1 was not toxic up to 10 µM and stifled IL-1ß-induced cyclooxygenase 2, prostaglandin E2, inducible nitric oxide synthase, nitric oxide, and matrix metalloproteinase-13. Our study of signalling pathways revealed that RvD1 suppressed IL-1ß-induced activation of NF-κB/p65, p38/MAPK and JNK(1/2). Moreover, RvD1 prevented HNE-induced cell apoptosis and oxidative stress, as indicated by inactivation of caspases, inhibition of lactate dehydrogenase release, and increased levels of Bcl2 and AKT, as well as GSH. CONCLUSION: This is the first in vitro study demonstrating the beneficial effect of RvD1 in OA. That RvD1 abolishing a number of factors known to be involved in OA pathogenesis renders it a clinically valuable agent in prevention of the disease.

Cellular Aging, Senescence and Autophagy Processes in Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis and a huge health and financial burden. The prevalence and incidence of OA are likely to rise due to increasing life expectancy. Although the link between aging and OA is well established, little is known about the mechanisms by which aging contributes to OA development. In recent years, progress has been made in understanding the molecular mechanisms of chondrocyte aging and senescence. Aging and senescent chondrocytes display a senescence-associated secretory phenotype (SASP) associated with increased secretion of pro-inflammatory mediators, extracellular matrix degrading enzymes and oxidative stress, all of which can contribute to the development and progression of OA. There is also evidence that autophagy, an essential homeostatic process, declines with aging and during OA. This review will focus on our current understanding of chondrocyte aging, senescence, and autophagy and their potential roles in the development and progression of OA. An understanding of these processes would be very useful in devising strategies to treat OA or to delay its development.

Covalent binding of 4-hydroxynonenal to matrix metalloproteinase 13 studied by liquid chromatography-mass spectrometry.

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 1ß-activated chondrocytes from OA patients using HR-MS/MS and MRM analysis.