Maria Isabel Guillén

Anti-Inflammatory Actions of the Heme Oxygenase-1 Pathway

Heme oxygenase 1 (HO-1) is induced by oxidative or nitrosative stress, cytokines and other mediators produced during inflammatory processes, likely as part of a defence system in cells exposed to stress to provide a negative feedback for cell activation and the production of mediators, which could modulate the inflammatory response. HO-1 activity results in the inhibition of oxidative damage and apoptosis, with significant reductions in inflammatory events including edema, leukocyte adhesion and migration, and production of inflammatory cytokines. HO-1 is induced by nitric oxide (NO) in different biological systems and can control the increased production of this mediator observed in many inflammatory situations. Regulatory interactions between HO-1 and cyclooxygenase (COX) pathways have also been reported. Modulation of signal transduction pathways by HO-1 or products derived from its activity, such as carbon monoxide (CO), may mediate the anti-inflammatory effects of this protein. Regulation of HO-1 activity may be a therapeutical strategy for a number of inflammatory conditions.

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.

High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1β in osteoarthritic synoviocytes.

IntroductionHigh mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).MethodsHMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-κB activation by transient transfection with a NF-κB-luciferase plasmid.ResultsIn the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1β, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-κB activation in the presence of IL-1β.ConclusionsOur results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1β to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.

Expression of heme oxygenase-1 and regulation by cytokines in human osteoarthritic chondrocytes

Heme oxygenase-1 (HO-1) is implicated in the protection against tissue injury. We investigated the expression of this protein in cartilage sections and chondrocytes obtained from osteoarthritic patients. HO-1 was immunodetected in preparations from cartilage and also in chondrocytes cultured in the absence of stimulation. We found that HO-1 can be modulated by cytokines since the pro-inflammatory cytokines interleukin (IL)-1beta, IL-17 and tumour necrosis factor-alpha (TNF-alpha) down-regulated this protein, whereas the anti-inflammatory cytokine IL-10 exerted the opposite effect. Our results suggest a role for HO-1 as part of protective mechanisms against tissue injury in human cartilage.

Haem oxygenase‐1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes

Pro‐inflammatory cytokines, matrix metalloproteinases (MMPs) and other catabolic factors participate in the pathogenesis of cartilage damage in osteoarthritis (OA). Pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) mediate cartilage degradation and might be involved in the progression of OA. Previously, we found that haem oxygenase‐1 (HO‐1) is down‐regulated by pro‐inflammatory cytokines and up‐regulated by IL‐10 in OA chondrocytes. The aim of this study was to determine whether HO‐1 can modify the catabolic effects of IL‐1β in OA cartilage and chondrocytes. Up‐regulation of HO‐1 by cobalt protoporphyrin IX significantly reduced glycosaminoglycan degradation elicited by IL‐1β in OA cartilage explants but increased glycosaminoglycan synthesis and the expression of collagen II in OA chondrocytes in primary culture, as determined by radiometric procedures, immunoblotting and immunocytochemistry. HO‐1 decreased the activation of extracellular signal‐regulated kinase 1/2. This was accompanied by a significant inhibition in MMP activity and expression of collagenases MMP‐1 and MMP‐13 at the protein and mRNA levels. In addition, HO‐1 induction caused a significant increase in the production of insulin‐like growth factor‐1 and a reduction in the levels of insulin‐like growth factor binding protein‐3. We have shown in primary culture of chondrocytes and articular explants from OA patients that HO‐1 counteracts the catabolic and anti‐anabolic effects of IL‐1β. Our data thus suggest that HO‐1 may be a factor regulating the degradation and synthesis of extracellular matrix components in OA. Copyright

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.

Carbon Monoxide-Releasing Molecules: A Pharmacological Expedient to Counteract Inflammation

Carbon monoxide (CO) mediates many of the biological effects that are attributed to heme oxygenase (HO), the enzyme responsible for CO production in mammals. Antioxidant and anti-inflammatory activities of HO-1, the inducible isoform of heme oxygenase, have been demonstrated in a variety of disease models and a therapeutic exploitation of this pathway is currently under scrutiny. In this context, the liberation of CO from CO-releasing molecules (CO-RMs) is extremely attractive as these compounds may form the basis of a new class of pharmaceuticals. Recent investigations indicate that HO-1 and CO modulate important processes in chronic inflammation; these include the control of immune responses, the production of inflammatory mediators and the mitigation of cartilage or bone destruction. As HO-1 is highly expressed in the joint tissues of patients affected by arthritic diseases, it is plausible to suggest that this pathway may play a protective role against joint degenerative diseases. Studies aimed at identifying the signaling pathways responsive to endogenous CO and CO-RMs in rheumatoid arthritis and other inflammatory states are currently in progress. This research will help to elucidate the molecular mechanisms underlying the pharmacological effects of CO-RMs and may lead to the development of novel therapeutic strategies for the treatment of acute and chronic inflammatory conditions.

Modulation of haem oxygenase-1 expression by nitric oxide and leukotrienes in zymosan-activated macrophages

Phagocytosis of unopsonized zymosan by RAW 264.7 macrophages upregulated protein expression of haem oxygenase‐1 (HO‐1), inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) in a time‐ and concentration‐dependent manner. In the presence of zymosan, exogenous prostaglandin E2 (PGE2) did not exert significant effects on the expression of these three enzymes. In contrast, exogenous leukotriene B4 (LTB4) and LTC4 in the nanomolar range inhibited HO‐1 and iNOS expression, as well as nitrite accumulation. The COX inhibitors indomethacin and NS398 weakly inhibited HO‐1 expression but had no effect on iNOS and COX‐2 expression or nitrite. In contrast, the 5‐lipoxygenase (5‐LO) inhibitor ZM 230,487 significantly decreased HO‐1, iNOS and nitrite, which were not affected by zileuton. Dexamethasone showed an inhibitory effect on HO‐1 expression induced by zymosan. ZM 230,487 but not zileuton, inhibited the shift due to nuclear factor‐κB (NF‐κB), whereas they did not modify activator protein‐1 (AP‐1) binding. Our results suggest that inhibition of NF‐κB binding could mediate the effects of ZM 230,487 on the modulation of HO‐1 and iNOS protein expression. NOS inhibition by L‐NG‐nitroarginine methyl ester (L‐NAME) or 1400 W abolished nitrite production and strongly reduced HO‐1 expression. These results show an induction of HO‐1 protein expression by zymosan phagocytosis in macrophages, with a positive modulatory role for endogenous NO and a negative regulation by exogenous LTs, likely dependent on the reduction of iNOS expression and NO production.