Fundamentals of osteoarthritis: Inflammatory mediators in osteoarthritis.

OBJECTIVES: As more has become known of the pathophysiology of osteoarthritis (OA), evidence that inflammation plays a critical role in its development and progression has accumulated. Here, we aim to review current knowledge of the complex inflammatory network in the OA joint. DESIGN: This narrative review is presented in three main sections: local inflammation, systemic inflammation, and therapeutic implications. We focused on inflammatory mediators and their link to OA structural changes in the joint. RESULTS: OA is characterized by chronic and low-grade inflammation mediated mostly by the innate immune system, which results in cartilage degradation, bone remodeling and synovial changes. Synovitis is regarded as an OA characteristic and associated with increased severity of symptoms and joint dysfunction. However, the articular cartilage and the subchondral bone also produce several pro-inflammatory mediators thus establishing a complex interplay between the different tissues of the joint. In addition, systemic low-grade inflammation induced by aging, obesity and metabolic syndrome can contribute to OA development and progression. The main inflammatory mediators associated with OA include cytokines, chemokines, growth factors, adipokines, and neuropeptides. CONCLUSIONS: Future research is needed to deeper understand the molecular pathways mediating the inflammation in OA to provide new therapeutics that target these pathways, or to repurpose existing drugs.

Innovation in Targeted Intra-articular Therapies for Osteoarthritis.

Osteoarthritis is the most common chronic joint disease characterized by progressive damage to the joints, leading to pain and loss of function. There is currently no cure or disease-modifying therapy for osteoarthritis. Hence, the increasing disease prevalence linked with ageing and obesity represents a substantial socio-economic burden. Intra-articular therapy by injection of drugs into affected joints can optimize local drug bioavailability, while reducing risks of systemic toxicity, a concern in an ageing patient population. In this review, we investigate the current landscape of intra-articular drug therapies for osteoarthritis, including established approaches and those in clinical development. We performed a literature review using PubMed, complemented with a search for clinical trials using the ClinicalTrials.gov repository. Additionally, conference abstracts and presentations were identified and systematic snowballing was applied. Identified drugs were divided into several groups by main mechanism of action, and include drugs that reduce inflammation (anti-inflammatory), drugs aiming to prevent or reverse structural damage (structure modifying), drugs that aim to reduce the pain, and other drugs with a specific target. Most studies have been performed for osteoarthritis of the knee, a joint that is easily accessible for intra-articular treatments. Optimal therapy would provide symptomatic relief, while preventing further damage to the joint. The field of intra-articular drug therapies for osteoarthritis is rapidly evolving with clear challenges identified: definition of relevant outcome measures, optimization of clinical trial set-ups, and dealing with placebo responses. While many uncertainties persist, it appears that the innovation in drug development and improved clinical trial set-up may finally deliver successful therapies for this important disease.

Inhibition of KDM7A/B histone demethylases restores H3K79 methylation and protects against osteoarthritis.

OBJECTIVES: In osteoarthritis, methylation of lysine 79 on histone H3 (H3K79me), a protective epigenetic mechanism, is reduced. Histone methylation levels are dynamically regulated by histone methyltransferases and demethylases. Here, we aimed to identify which histone demethylases regulate H3K79me in cartilage and investigate whether their targeting protects against osteoarthritis. METHODS: We determined histone demethylase expression in human non-osteoarthritis and osteoarthritis cartilage using qPCR. The role of histone demethylase families and subfamilies on H3K79me was interrogated by treatment of human C28/I2 chondrocytes with pharmacological inhibitors, followed by western blot and immunofluorescence. We performed C28/I2 micromasses to evaluate effects on glycosaminoglycans by Alcian blue staining. Changes in H3K79me after destabilisation of the medial meniscus (DMM) in mice were determined by immunohistochemistry. Daminozide, a KDM2/7 subfamily inhibitor, was intra-articularly injected in mice upon DMM. Histone demethylases targeted by daminozide were individually silenced in chondrocytes to dissect their role on H3K79me and osteoarthritis. RESULTS: We documented the expression signature of histone demethylases in human non-osteoarthritis and osteoarthritis articular cartilage. Inhibition of Jumonji-C demethylase family increased H3K79me in human chondrocytes. Blockade of KDM2/7 histone demethylases with daminozide increased H3K79me and glycosaminoglycans. In mouse articular cartilage, H3K79me decayed rapidly upon induction of joint injury. Early and sustained intra-articular treatment with daminozide enhanced H3K79me and exerted protective effects in mice upon DMM. Individual silencing of KDM7A/B demethylases in human chondrocytes demonstrated that KDM7A/B mediate protective effects of daminozide on H3K79me and osteoarthritis. CONCLUSION: Targeting KDM7A/B histone demethylases could be an attractive strategy to protect joints against osteoarthritis.

Hypoxia induces DOT1L in articular cartilage to protect against osteoarthritis.

Osteoarthritis is the most prevalent joint disease worldwide, and it is a leading source of pain and disability. To date, this disease lacks curative treatment, as underlying molecular mechanisms remain largely unknown. The histone methyltransferase DOT1L protects against osteoarthritis, and DOT1L-mediated H3K79 methylation is reduced in human and mouse osteoarthritic joints. Thus, restoring DOT1L function seems to be critical to preserve joint health. However, DOT1L-regulating molecules and networks remain elusive, in the joint and beyond. Here, we identified transcription factors and networks that regulate DOT1L gene expression using a potentially novel bioinformatics pipeline. Thereby, we unraveled a possibly undiscovered link between the hypoxia pathway and DOT1L. We provide evidence that hypoxia enhanced DOT1L expression and H3K79 methylation via hypoxia-inducible factor-1 α (HIF1A). Importantly, we demonstrate that DOT1L contributed to the protective effects of hypoxia in articular cartilage and osteoarthritis. Intra-articular treatment with a selective hypoxia mimetic in mice after surgical induction of osteoarthritis restored DOT1L function and stalled disease progression. Collectively, our data unravel a molecular mechanism that protects against osteoarthritis with hypoxia inducing DOT1L transcription in cartilage. Local treatment with a selective hypoxia mimetic in the joint restores DOT1L function and could be an attractive therapeutic strategy for osteoarthritis.

Promising targets for therapy of osteoarthritis: a review on the Wnt and TGF-ß signalling pathways.

Osteoarthritis (OA) is the most common chronic joint disorder worldwide, with a high personal burden for the patients and an important socio-economic impact. Current therapies are largely limited to pain management and rehabilitation and exercise strategies. For advanced cases, joint replacement surgery may be the only option. Hence, there is an enormous need for the development of effective and safe disease-modifying anti-OA drugs. A strong focus in OA research has been on the identification and role of molecular signalling pathways that contribute to the balance between anabolism and catabolism in the articular cartilage. In this context, most insights have been gained in understanding the roles of the transforming growth factor-beta (TGF-ß) and the Wingless-type (Wnt) signalling cascades. The emerging picture demonstrates a high degree of complexity with context-dependent events. TGF-ß appears to protect cartilage under healthy conditions, but shifts in its receptor use and subsequent downstream signalling may be deleterious in aged individuals or in damaged cartilage. Likewise, low levels of Wnt activity appear important to sustain chondrocyte viability but excessive activation is associated with progressive joint damage. Emerging clinical data suggest some potential for the use of sprifermin, a recombinant forms of fibroblast growth factor 18, a distant TGF-ß superfamily member, and for lorecivivint, a Wnt pathway modulator.

Review Article: Is Wnt Signaling an Attractive Target for the Treatment of Osteoarthritis?

Osteoarthritis is the most common chronic joint disease affecting millions of people worldwide and a leading cause of pain and disability. Increasing incidence of obesity and aging of the population are two factors that suggest that the impact of osteoarthritis will further increase at the society level. Currently, there are no drugs available that can manage both structural damage to the joint or the associated pain. Increasing evidence supports the view that the Wnt signaling pathway plays an important role in this disease. The current concept, based on genetic and functional studies, indicates that tight regulation of Wnt signaling in cartilage is essential to keep the joint healthy. In this review, we discuss how this concept has evolved, provide insights into the regulation of Wnt signaling, in particular by Wnt modulators such as frizzled-related protein and DOT1-like histone lysine methyltransferase, and summarize preclinical evidence and molecular mechanisms of lorecivivint, the first Wnt antagonist in clinical development for osteoarthritis.

ANP32A regulates ATM expression and prevents oxidative stress in cartilage, brain, and bone.

Osteoarthritis is the most common joint disorder with increasing global prevalence due to aging of the population. Current therapy is limited to symptom relief, yet there is no cure. Its multifactorial etiology includes oxidative stress and overproduction of reactive oxygen species, but the regulation of these processes in the joint is insufficiently understood. We report that ANP32A protects the cartilage against oxidative stress, preventing osteoarthritis development and disease progression. ANP32A is down-regulated in human and mouse osteoarthritic cartilage. Microarray profiling revealed that ANP32A protects the joint by promoting the expression of ATM, a key regulator of the cellular oxidative defense. Antioxidant treatment reduced the severity of osteoarthritis, osteopenia, and cerebellar ataxia features in Anp32a-deficient mice, revealing that the ANP32A/ATM axis discovered in cartilage is also present in brain and bone. Our findings indicate that modulating ANP32A signaling could help manage oxidative stress in cartilage, brain, and bone with therapeutic implications for osteoarthritis, neurological disease, and osteoporosis.

SMOC2 inhibits calcification of osteoprogenitor and endothelial cells.

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2's inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders.

Cushioning the cartilage: a canonical Wnt restricting matter.

Wnt signalling pathways have key roles in joint development, homeostasis and disease, particularly in osteoarthritis. New data is starting to reveal the importance of tightly regulating canonical Wnt signalling pathway activation to maintain homeostasis and health in articular cartilage. In addition to the presence of different Wnt antagonists that limit pathway activation in articular cartilage, the reciprocal crosstalk between the canonical and non-canonical cascades and competitive antagonism between different Wnt ligands seem to be critical in restraining excessive Wnt pathway activation. Changes in transcriptional complex assembly upon Wnt pathway activation, epigenetic modulation of target gene transcription, in particular through histone modifications, and complex interactions between the Wnt signalling pathway and other signalling pathways, are also instrumental in adjusting Wnt signalling. In this Review, the cellular and molecular mechanisms involved in fine-tuning canonical Wnt signalling in the joint are updated, with a focus on the articular cartilage. The interventions for preventing or treating osteoarthritis are also discussed, which should aim to limit disease-associated excessive canonical Wnt activity to avoid joint damage.

DOT1L safeguards cartilage homeostasis and protects against osteoarthritis.

Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis.

Suramin increases cartilage proteoglycan accumulation in vitro and protects against joint damage triggered by papain injection in mouse knees in vivo.

OBJECTIVES: Suramin is an old drug used for the treatment of African sleeping sickness. We investigated therapeutic repositioning of suramin to protect against cartilage damage, as suramin may interact with tissue inhibitor of metalloproteinase-3 (TIMP3). METHODS: In vitro extracellular matrix (ECM) accumulation and turnover in the presence or absence of suramin were studied in the ATDC5 micromass model of chondrogenesis and in pellet cultures of human articular chondrocytes from osteoarthritis and control patients, by gene expression, protein analysis, colorimetric staining, immunoprecipitation, fluorimetric analysis and immunohistochemistry. To study suramin in vivo, the drug was injected intra-articularly in the papain model of joint damage. Disease severity was analysed by histology, immunohistochemistry and contrast-enhanced nanofocus CT. RESULTS: In ATDC5 micromasses, suramin increased TIMP3 levels and decreased the activity of matrix metalloproteinases (MMPs) and aggrecanases. Suramin treatment resulted in increased glycosaminoglycans. This effect on the ECM was blocked by an anti-TIMP3 antibody. Direct interaction between suramin and endogenous TIMP3 was demonstrated in immunoprecipitates. Mice treated intra-articularly with suramin injections showed reduced cartilage damage compared with controls, with increased TIMP3 and decreased MMP and aggrecanase activity. Translational validation in human chondrocytes confirmed increased TIMP3 function and reduced cartilage breakdown after suramin treatment. CONCLUSION: Suramin prevented loss of articular cartilage in a mouse model of cartilage damage. The effects appear to be mediated by a functional increase of TIMP3 and a subsequent decrease in the activity of catabolic enzymes. Thus, suramin repositioning could be considered to prevent progressive cartilage damage and avoid evolution toward osteoarthritis.

Wnt signaling as target for the treatment of osteoarthritis.

Osteoarthritis is a severe and common rheumatic and skeletal disease for which currently no specific drugs are available. The Wnt signaling pathway modulates key biological processes in development, growth, homeostasis, and disease, particularly in the joints and bone. Excessive activation of the Wnt signaling pathway in the articular cartilage has been clearly associated with the onset and severity of osteoarthritis. Hence, targeting Wnt signaling may be an excellent approach to develop specific drugs useful for the treatment of osteoarthritis. In this article, we review the biology of Wnt signaling in the context of osteoarthritis; we also analyze the gradual improvement of our molecular understanding of Wnts in the joint and oversee current progress toward the development of Wnt inhibition as therapy for osteoarthritis. At least one Wnt inhibitor is currently going forward in the clinical evaluation process, potentially marking the beginning of a new era in the management of osteoarthritis.

Nanoparticles for brain-specific drug and genetic material delivery, imaging and diagnosis.

The poor access of therapeutic drugs and genetic material into the central nervous system due to the presence of the blood-brain barrier often limits the development of effective noninvasive treatments and diagnoses of neurological disorders. Moreover, the delivery of genetic material into neuronal cells remains a challenge because of the intrinsic difficulty in transfecting this cell type. Nanotechnology has arisen as a promising tool to provide solutions for this problem. This review will cover the different approaches that have been developed to deliver drugs and genetic material efficiently to the central nervous system as well as the main nanomaterials used to image the central nervous system and diagnose its disorders.

Inhibition of p42 MAPK using a nonviral vector-delivered siRNA potentiates the anti-tumor effect of metformin in prostate cancer cells.

AIMS: The aim of this work was to study if a G1-polyamidoamine dendrimer/siRNA dendriplex can remove the p42 MAPK protein in prostate cancer cells and to potentiate the anti-tumoral effect of the antidiabetic drug metformin and taxane docetaxel. MATERIAL & METHODS: The dendriplex uptake was studied using flow cytometry analysis. Transfection efficiency was determined by measuring p42 MAPK mRNA and protein levels. Anti-tumoral effects were determined by measuring cellular proliferation and damage. RESULTS: The dendriplex siRNA/G1-polyamidoamine dendrimer decreased both p42 MAPK mRNA and protein levels by more than 80%, which potentiates the anti-tumoral effects of metformin. CONCLUSION: Blockade of the MAPK pathway using a dendrimer-vehiculized siRNA to block the MAPK signaling pathway in prostate cancer cells can potentiate the anti-tumoral activity of anticancer drugs, indicating that the combination of siRNA-mediated blockade of survival signals plus anti-tumoral therapy might be a useful approach for cancer therapy.

Efficient, non-toxic hybrid PPV-PAMAM dendrimer as a gene carrier for neuronal cells.

A novel hybrid dendrimer (TRANSGEDEN) that combines a conjugated rigid polyphenylenevinylene (PPV) core with flexible polyamidoamine (PAMAM) branches at the surface was synthesized and characterized. The potential of this material as a nonviral gene delivery system was also examined, and it was observed that dendriplexes formed by TRANSGEDEN and small interfering ribonucleic acids (siRNAs) can be incorporated into >90% of neuronal cells without any toxicity up to a dendrimer concentration of 3 µM. TRANSGEDEN was used to deliver a specific siRNA to rat cerebellar granular neurons (CGNs) to knock down the cofilin-1 protein. Cofilin-1 removal partially protects CGNs from N-methyl D-aspartate (NMDA)-mediated neuronal death.

Atorvastatin reduces high glucose toxicity in rat peritoneal mesothelial cells.

OBJECTIVE: Continuous exposure of the peritoneal membrane to high glucose dialysis solutions can produce functional alterations in this membrane. We studied the toxic effects of high glucose (50 mmol/L and 83 mmol/L) and its reversal by atorvastatin (0.5 - 5 µmol/L) on cultures of rat peritoneal mesothelial cells (PMCs). METHODS: Rat PMCs were harvested from the peritonea of male Sprague-Dawley rats and grown in M199 medium supplemented with 10% fetal bovine serum. The effects of high glucose (50 mmol/L and 83 mmol/L) on levels of reactive oxygen species (ROS), on caspase 3 activity, and on phospho-p38 mitogen-activated protein kinase (MAPK) in the cultures were evaluated. RESULTS: Exposure to high glucose (for 4, 8, and 24 hours) increased intracellular levels of ROS and phospho-p38 MAPK (indices of cellular toxicity). Atorvastatin blocked these toxic effects of high glucose, being more effective against 50 mmol/L glucose (protective effects were observed above 0.5 µmol/L) than against 83 mmol/L (protective effects were observed above 2.5 µmol/L). Atorvastatin was also able to prevent glucose-induced increase in caspase 3 activity. CONCLUSIONS: The present study shows that high glucose may promote oxidative stress and may activate apoptotic pathways in rat PMCs. These toxic effects could be reversed by atorvastatin.

N-sulfinyl amines as a nitrogen source in the asymmetric intramolecular aza-Michael reaction: total synthesis of (-)-pinidinol.

N-Sulfinyl amines have been successfully employed as nitrogen nucleophiles for the asymmetric intramolecular aza-Michael reaction. The synthetic strategy involves a cross-metathesis reaction followed by the Michael-type cyclization, either in a base-catalyzed two-step procedure or in a tandem fashion. The developed methodology allows access to chiral substituted pyrrolidines and piperidines bearing one or two stereocenters and it has been applied to the synthesis of the piperidine alkaloid (-)-pinidinol.

Asymmetric synthesis of indolines through intramolecular shifting of aromatic sulfinyl groups. Role of the pi,pi-stacking interactions in these unusual S(N)Ar processes.

Cyclization of N-aryl substituted 1-aryl-2[2-p-tolylsulfinyl]phenyl propylamines under LDA, LHMDS, or KHMDS provides a new approach for synthesizing optically pure 2,3-disubstituted indolines. Both the scope and the limitations of this method have been investigated. The pi,pi-stacking interactions are crucial for these unprecedented intramolecular S(N)Ar processes, in which a sulfinyl group located on a slightly deactivated ring is substituted by amide anions under mild conditions. X-ray and NMR proofs supporting these pi,pi-stacking interactions are presented.