Javier Megías

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.

The carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cells.

Recent evidence indicates that carbon monoxide‐releasing molecules (CO‐RMs) exhibit potential anti‐inflammatory properties. In the present study, we have investigated whether tricarbonyl dichloro ruthenium(II) dimer (CORM‐2) can control the inflammatory response induced by cytokines in a human colonic epithelial cell line, Caco‐2.

Direct Toll-like receptor-mediated stimulation of hematopoietic stem and progenitor cells occurs in vivo and promotes differentiation toward macrophages.

As Toll‐like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl‐S‐glyceryl‐L‐Cys‐Ser‐(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin−) as well as HSPCs (identified as Lin− c‐Kit+ Sca‐1+ IL‐7Rα− [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)‐dependent pathway. In order to investigate the possible direct interaction of soluble microorganism‐associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin− and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2−/−, TLR4−/−, or MyD88−/− mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen‐derived products directly during infection, inducing a rapid generation of cells of the innate immune system. STEM CELLS2012;30:1486–1495

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

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.

Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin− c-Kit+ Sca-1+ IL-7Rα−), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2−/− mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2−/− mice correlating with their higher fungal burden. Accordingly, emigration of Ly6Chigh monocytes and neutrophils to spleen was increased in TLR2−/− mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2−/− infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin− cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.

The Carbon Monoxide-Releasing Molecule Tricarbonyldichlororuthenium(II) Dimer Protects Human Osteoarthritic Chondrocytes and Cartilage from the Catabolic Actions of Interleukin-1β

We have investigated the effects of a carbon monoxide-releasing molecule, tricarbonyldichlororuthenium(II) dimer (CORM-2), on catabolic processes in human osteoarthritis (OA) cartilage and chondrocytes activated with interleukin-1β. In these cells, proinflammatory cytokines induce the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, including members of a disintegrin and metalloproteinase with thrombospondin domain (ADAMTS) family, which may contribute to cartilage loss. CORM-2 down-regulated MMP-1, MMP-3, MMP-10, MMP-13, and ADAMTS-5 in OA chondrocytes, and it inhibited cartilage degradation. These effects were accompanied by increased aggrecan synthesis and collagen II expression in chondrocytes. Our results also indicate that the inhibition of extracellular signal-regulated kinase 1/2 and p38 activation by CORM-2 may contribute to the maintenance of extracellular matrix homeostasis. These observations suggest that CORM-2 could exert chondroprotective effects due to the inhibition of catabolic activities and the enhancement of aggrecan synthesis.

Retinal microglia are activated by systemic fungal infection

PURPOSE We determined whether systemic fungal infection could cause activation of retinal microglia and, therefore, could be potentially harmful for patients with retinal degenerative diseases. METHODS Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by confocal immunofluorescence and flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII, and anti-CD45 antibodies. RESULTS Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial relocation in retinal layers. CONCLUSIONS As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration, or retinitis pigmentosa.