Victoria Clérigues

New molecular targets for the treatment of osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by destruction of the articular cartilage, subchondral bone alterations and synovitis. Current treatments are focused on symptomatic relief but they lack efficacy to control the progression of this disease which is a leading cause of disability. Therefore, the development of effective disease-modifying drugs is urgently needed. Different initiatives are in progress to define the molecular mechanisms involved in the initiation and progression of OA. These studies support the therapeutic potential of pathways relevant in joint metabolism such as Wnt/beta-catenin, discoidin domain receptor 2 or proteinase-activated receptor 2. The dysregulation in cartilage catabolism and subchondral bone remodeling could be improved by selective inhibitors of matrix metalloproteinases, aggrecanases and other proteases. Another approach would favor the activity of anabolic processes by using growth factors or regulatory molecules. Recent studies have also revealed the role of oxidative stress and synovitis in the progression of this disease, supporting the development of a number of inhibitory strategies. Novel targets in OA are represented by genes involved in OA pathophysiology discovered using gene network, epigenetic and microRNA approaches. Further insights into the molecular mechanisms involved in OA initiation and progression may lead to the development of new therapies able to control joint destruction and repair.

Heme oxygenase-1 mediates protective effects on inflammatory, catabolic and senescence responses induced by interleukin-1β in osteoarthritic osteoblasts

Osteoarthritis (OA) is a chronic degenerative joint disease showing altered bone metabolism. Osteoblasts contribute to the regulation of cartilage metabolism and bone remodeling. We have shown previously that induction of heme oxygenase-1 (HO-1) protects OA cartilage against inflammatory and degradative responses. In this study, we investigated the effects of HO-1 induction on OA osteoblast metabolism. HO-1 was induced with cobalt protoporphyrin IX (CoPP) and by transduction with LV-HO-1. In osteoblasts stimulated with interleukin (IL)-1β, CoPP enhanced mineralization, the expression of a number of markers of osteoblast differentiation such as Runx2, bone morphogenetic protein-2, osteocalcin, and collagen 1A1 and 1A2, as well as the ratio osteoprotegerin/receptor activator of nuclear factor-κB ligand. HO-1 induction significantly reduced the expression of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-3, and the production of pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6 whereas IL-10 levels increased. HO-1 also exerted inhibitory effects on prostaglandin (PG)E(2) production which could be dependent on cyclooxygenase-2 and microsomal PGE synthase-1 down-regulation. The activity of senescence-associated β-galactosidase and the expression of the senescence marker caveolin-1 were significantly decreased after HO-1 induction. The inhibition of nuclear factor-κB activation induced by IL-1β in OA osteoblasts may contribute to some HO-1 effects. Our results have shown that HO-1 decreases the production of relevant inflammatory and catabolic mediators that participate in OA pathophysiology thus eliciting protective effects in OA osteoblasts.

The CO-releasing molecule CORM-2 is a novel regulator of the inflammatory process in osteoarthritic chondrocytes

OBJECTIVES Previous work has shown that the CO-releasing molecule CORM-2 protects against cartilage degradation. The aim of this study was to examine whether CORM-2 can control the production of inflammatory mediators in osteoarthritic chondrocytes and determine the mechanisms involved. METHODS Primary cultures of chondrocytes from OA patients were stimulated with IL-1beta. The production of reactive oxygen species, nitrite, PGE(2), TNF-alpha and IL-1 receptor antagonist (IL-1Ra) were measured in the presence or absence of CORM-2. The expression of nitric oxide synthase-2 (NOS-2), cyclo-oxygenase-2 (COX-2) and microsomal PG E synthase-1 (mPGES-1) was followed by western blot and real-time PCR. Activation of nuclear factor-kappaB (NF-kappaB) and hypoxia inducible factor-1alpha (HIF-1alpha), and phosphorylation of NF-kappaB inhibitory protein alpha (IkappaBalpha) were determined by ELISA. RESULTS CORM-2 decreased the production of oxidative stress, nitrite and PGE(2). In addition, CORM-2 inhibited IL-1beta-induced TNF-alpha but enhanced IL-1Ra production. Treatment of chondrocytes with CORM-2 strongly down-regulated NOS-2 and mPGES-1 protein expression, whereas COX-2 was reduced to a lesser extent. These changes were accompanied by a significant decrease in mRNA expression for NOS-2 and mPGES-1. CORM-2 showed a concentration-dependent inhibition of DNA-binding activity for p65 NF-kappaB and HIF-1alpha. IkappaBalpha phosphorylation was also reduced by CORM-2 treatment. CONCLUSIONS These data have opened new mechanisms of action for CORM-2, raising the prospect that CO-releasing molecules are an interesting strategy for the development of new treatments in articular conditions.

Heme oxygenase-1 induction modulates microsomal prostaglandin E synthase-1 expression and prostaglandin E2 production in osteoarthritic chondrocytes

Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) may participate in the pathogenesis of cartilage damage in osteoarthritis (OA) through the production of catabolic enzymes and inflammatory mediators. Induction of heme oxygenase-1 (HO-1) has previously been shown to exert anti-inflammatory effects in different cell types. We have investigated whether HO-1 induction may modify chondrocyte viability and the production of relevant mediators such as oxidative stress and prostaglandin E(2) (PGE(2)) elicited by IL-1beta in OA chondrocytes. Chondrocytes were isolated from OA cartilage and used in primary culture. Cells were stimulated with IL-1beta in the absence or presence of the HO-1 inducer cobalt protoporphyrin IX (CoPP). Gene expression was assessed by quantitative real-time PCR, protein levels by ELISA and Western blot, apoptosis by laser scanning cytometry using annexin V-FITC and TUNEL assays, and oxidative stress by LSC with dihydrorhodamine 123. HO-1 induction by CoPP enhanced chondrocyte viability and aggrecan content while inhibiting apoptosis and oxidative stress generation. PGE(2) is produced in OA chondrocytes stimulated by IL-1beta by the coordinated induction of cyclooxygenase-2 and microsomal PGE synthase 1 (mPGES-1). The production of PGE(2) was decreased by HO-1 induction as a result of diminished mPGES-1 protein and mRNA expression. Transfection with HO-1 small interfering RNA counteracted CoPP effects. In addition, the activation of nuclear factor-kappaB and early growth response-1 was significantly reduced by CoPP providing a basis for its anti-inflammatory effects. These results confirm the protective role of HO-1 induction in OA chondrocytes and suggest the potential interest of this strategy in degenerative joint diseases.

Haem oxygenase-1 counteracts the effects of interleukin-1β on inflammatory and senescence markers in cartilage-subchondral bone explants from osteoarthritic patients.

IL (interleukin)-1β plays an important role in cartilage extracellular matrix degradation and bone resorption in OA (osteoarthritis) through the induction of degradative enzymes and pro-inflammatory mediators. In the present study, we have determined the consequences of HO-1 (haem oxygenase-1) induction on markers of inflammation and senescence in the functional unit cartilage-subchondral bone stimulated with IL-1β. Cartilage-subchondral bone specimens were obtained from the knees of osteoarthritic patients. Treatment with the HO-1 inducer CoPP (cobalt protoporphyrin IX) counteracted the stimulatory effects of IL-1β on IL-6, nitrite, PGE2 (prostaglandin E2), TGF (transforming growth factor) β2, TGFβ3 and osteocalcin. Immunohistochemical analyses indicated that CoPP treatment of explants down-regulated iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase-2) and mPGES-1 (microsomal prostaglandin E synthase-1) induced by IL-1β. In contrast, the expression of HMGB1 (high-mobility group box 1) was not significantly modified. In addition, CoPP decreased the expression of iNOS and mPGES-1 in cells isolated from the explants and stimulated with IL-1β, which was counteracted by an siRNA (small interfering RNA) specific for human HO-1. In isolated primary chondrocytes, we determined senescence-associated β-galactosidase activity and the expression of senescence markers by real-time PCR. We have found that HO-1 induction could regulate senescence markers in the presence of IL-1β and significantly affected telomerase expression, as well as β-galactosidase activity and hTERT (human telomerase reverse transcriptase) and p21 expression in chondrocytes. The findings of the present study support the view that HO-1 induction results in the down-regulation of inflammatory and senescence responses in OA articular tissues.

Antioxidant and Antiinflammatory Properties of Heme Oxygenase-1 in Osteoarthritic Articular Cells

Heme oxygenase-1 (HO-1) is induced in cells by various stimuli as a defense system against oxidative stress. It is known that reactive oxygen species (ROS) participates in the initiation and progression of osteoarthritis (OA) and several antioxidant systems may protect cartilage components. HO-1 induction or CO release from CORM-2 counteracts oxidative stress and protects against proinflammatory and catabolic effects of interleukin-1β in OA chondrocytes, osteoblasts, and synoviocytes as well as in OA osteochondral explants. Both approaches have been able to downregulate the production of mediators such as reactive oxygen species, nitric oxide, matrix metalloproteinases, prostaglandin E2, cytokines, or chemokines accompanied by inhibition of cartilage degradation and improved aggrecan synthesis. Therefore, HO-1 or CO would be active on cell metabolism alterations, cartilage degradation, and synovitis. Understanding the mechanisms responsible for these effects may lead to novel strategies to prevent or treat joint destruction.

232 HEME OXYGENASE-1 REGULATES INFLAMMATORY MEDIATORS IN CARTILAGE ADJACENT TO SUBCHONDRAL BONE FROM OSTEOARTHRITIC PATIENTS

the adipokines to their receptors. Interestingly, the downregulation of adipokines was associated with the reduced expression of the cartilage-specific transcription factor Sox9 and a strong increase in the transcript level of collagen type 1. Chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in alginate beads, but ceased expressing their receptors. Conclusions: The modulation of chondrocyte phenotype induced by experimental conditions affects the expression of adipokines and their receptors. These experiment-dependent changes could result in modifications of cell response to leptin or adiponectin, and could therefore contribute to the discrepancies found in different studies. These findings suggest also that adipokines may play an essential role to prevent a phenotypic loss of chondrocyte function.