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.

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.

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.

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.

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.

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.

Sorbitol-modified hyaluronic acid reduces oxidative stress, apoptosis and mediators of inflammation and catabolism in human osteoarthritic chondrocytes.

OBJECTIVE AND DESIGN: Our study was designed to elucidate the precise molecular mechanisms by which sorbitol-modified hyaluronic acid (HA/sorbitol) exerts beneficial effects in osteoarthritis (OA). METHODS: 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-1ß) or hydrogen peroxide (H2O2). Signal transduction pathways and parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. RESULTS: HA/sorbitol prevented IL-1ß-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-1ß-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-1ß-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. CONCLUSIONS: 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.

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.

Inhibition of inducible nitric oxide synthase prevents lipid peroxidation in osteoarthritic chondrocytes.

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-1ß) or HNE at cytotoxic or non-cytotoxic concentrations. Parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. L-NIL stifled IL-1ß-induced NO release, iNOS activity, nitrated proteins, and HNE generation in a dose-dependent manner. It also blocked IL-1ß-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-1ß-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.

Contribution of H3K4 methylation by SET-1A to interleukin-1-induced cyclooxygenase 2 and inducible nitric oxide synthase expression in human osteoarthritis chondrocytes.

OBJECTIVE: To investigate the role of histone H3 lysine 4 (H3K4) methylation in interleukin-1ß (IL-1ß)-induced cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in human osteoarthritic (OA) chondrocytes. METHODS: Chondrocytes were stimulated with IL-1, and the expression of iNOS and COX-2 messenger RNA and proteins was evaluated by real-time reverse transcriptase-polymerase chain reaction analysis and Western blotting, respectively. H3K4 methylation and the recruitment of the histone methyltransferases SET-1A and MLL-1 to the iNOS and COX-2 promoters were evaluated using chromatin immunoprecipitation assays. The role of SET-1A was further evaluated using the methyltransferase inhibitor 5'-deoxy-5'-(methylthio)adenosine (MTA) and gene silencing experiments. SET-1A level in cartilage was determined using immunohistochemistry. RESULTS: The induction of iNOS and COX-2 expression by IL-1 was associated with H3K4 di- and trimethylation at the iNOS and COX-2 promoters. These changes were temporally correlated with the recruitment of the histone methyltransferase SET-1A, suggesting an implication of SET-1A in these modifications. Treatment with MTA inhibited IL-1-induced H3K4 methylation as well as IL-1-induced iNOS and COX-2 expression. Similarly, SET-1A gene silencing with small interfering RNA prevented IL-1-induced H3K4 methylation at the iNOS and COX-2 promoters as well as iNOS and COX-2 expression. Finally, we showed that the level of SET-1A expression was elevated in OA cartilage as compared with normal cartilage. CONCLUSION: These results indicate that H3K4 methylation by SET-1A contributes to IL-1-induced iNOS and COX-2 expression and suggest that this pathway could be a potential target for pharmacologic intervention in the treatment of OA and possibly other arthritic diseases.

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.

Nuclear receptor retinoid-related orphan receptor α1 modulates the metabolic activity of human osteoblasts.

Nuclear receptor retinoid-related orphan receptor alpha (RORα1) is a member of ROR-family receptors. It is broadly expressed in various tissues and organs during embryonic development. However, so far, little is known about its function in bone. Here, we have elucidated the expression and function of RORα1 in human MG-63 osteoblast-like cells. Reverse transcriptase-polymerase chain reaction and immunocytochemical analysis revealed that human MG-63 osteoblasts expressed and produced RORα1. Other cell lines, such as THP-1 monocytes expressed also RORα1. RORα1 over-expression increased alkaline phosphatase, osteocalcin, cell mineralization, and collagen type I mRNA and protein expression, while RORα1 RNA silencing inhibited these responses. In addition, RORα1 over-expression suppressed the tumor necrosis factor-alpha (TNFα)-induced production of cyclooxygenase-2, prostaglandin E(2) , and metalloproteinase-9. Examination of the signaling pathways disclosed that RORα1 was able to block TNFα-evoked nuclear factor-kappaB activation. In conclusion, this study demonstrates that RORα1 is involved in human osteoblast metabolism by stimulating osteoblast marker expression and inhibiting inflammatory responses. The results may encourage further exploration of RORα1 as a potential target for the treatment of bone disorders related to inflammation.

Elucidation of molecular mechanisms underlying the protective effects of thymoquinone against rheumatoid arthritis.

Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. A few studies have shown that TQ exhibits anti-inflammatory activities in experimental models of rheumatoid arthritis (RA) through mechanisms that are not fully understood. The aim of this work was to evaluate the in vitro and in vivo effects of TQ and to investigate its influence on the major signalling pathways involved in pathophysiological RA changes. We used isolated human RA fibroblast-like synoviocytes (FLS) and a rat adjuvant-induced arthritis model of RA. In isolated RA FLS, TQ (0-10 µM) was not cytotoxic and inhibited slightly lipopolysaccharide (LPS)-induced FLS proliferation and strongly H(2)O(2)-induced 4-hydroxynonenal (HNE) generation. By studying different inflammatory and catabolic factors, we determined that TQ significantly abolished LPS-induced interleukin-1beta (IL-1ß), tumour necrosis factor-alpha (TNFα), metalloproteinase-13, cyclooxygenase-2, and prostaglandin E(2). Furthermore, LPS-induced the phosphorylation of p38 mitogen-activated protein kinase, extracellular-regulated kinases ½, and nuclear factor-kappaB-p65 were also blocked by TQ in time-dependent manner. In our experimental RA model, the oral administration of TQ 5 mg/kg/day significantly reduced the serum levels of HNE, IL-1ß and TNFα as well as bone turnover markers, such as alkaline phosphatase and tartrate-resistant acid phosphatase. The protective effects of TQ against RA were also evident from the decrease in arthritis scoring and bone resorption. In conclusion, the fact that TQ abolishes a number of factors known to be involved in RA pathogenesis renders it a clinically valuable agent in the prevention of articular diseases, including RA.

Hydrodynamic delivery of chitosan-folate-DNA nanoparticles in rats with adjuvant-induced arthritis.

50 kDa chitosan was conjugated with folate, a specific tissue-targeting ligand. Nanoparticles such as chitosan-DNA and folate-chitosan-DNA were prepared by coacervation process. The hydrodynamic intravenous injection of nanoparticles was performed in the right posterior paw in normal and arthritic rats. Our results demonstrated that the fluorescence intensity of DsRed detected was 5 to 12 times more in the right soleus muscle and in the right gastro muscle than other tissue sections. ß-galactosidase gene expression with X-gal substrate and folate-chitosan-plasmid nanoparticles showed best coloration in the soleus muscle. Treated arthritic animals also showed a significant decrease in paw swelling and IL-1ß and PGE2 concentration in serum compared to untreated rats. This study demonstrated that a nonviral gene therapeutic approach using hydrodynamic delivery could help transfect more efficiently folate-chitosan-DNA nanoparticles in vitro/in vivo and could decrease inflammation in arthritic rats.

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.

Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury.

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia-reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia-reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.

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.