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IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKβ KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1β stimulated conditions. We find that in their response to IL-1β stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1β dependent increases. Our results suggest that IKKα could be a novel OA disease target.

During osteoarthritis development, chondrocytes are subjected to a functional derangement. This increases their susceptibility to stressful conditions such as oxidative stress, a characteristic of the aging tissue, which can further provoke extrinsic senescence by DNA damage responses. It was previously observed that IκB kinase α knockdown increases the replicative potential of primary human OA chondrocytes cultured in monolayer and the survival of the same cells undergoing hypertrophic-like differentiation in 3-D. In this paper we investigated whether IKKα knockdown could modulate oxidative stress-induced senescence of OA chondrocytes undergoing a DDR and particularly the involvement in this process of the DNA mismatch repair system, the principal mechanism for repair of replicative and recombinational errors, devoted to genomic stability maintenance in actively replicating cells. This repair system is also implicated in oxidative stress-mediated DNA damage repair. We analyzed microsatellite instability and expression of the mismatch repair components in human osteoarthritis chondrocytes after IKKα knockdown and H2O2 exposure. Only low MSI levels and incidence were detected and exclusively in IKKα proficient cells. Moreover, we found that IKKα proficient and deficient chondrocytes differently regulated MMR proteins after oxidative stress, both at mRNA and protein level, suggesting a reduced susceptibility of IKKα deficient cells. Our data suggest an involvement of the MMR system in the response to oxidative stress that tends to be more efficient in IKKαKD cells. This argues for a partial contribution of the MMR system to the better ability to recover DNA damage already observed in these cells.

Oxidative stress (OS) contributes to Osteoarthritis (OA) pathogenesis and its effects are worsened by the impairment of homeostatic mechanisms such as autophagy in OA chondrocytes. Rescue of an efficient autophagic flux could therefore reduce the bulk of damaged molecules, and at the same time improve cell function and viability. As a promising dietary or intra-articular supplement to rescue autophagy in OA chondrocytes, we tested spermidine (SPD), known to induce autophagy and to reduce OS in several other cellular models. Chondrocytes were obtained from OA cartilage and seeded at high-density to keep their differentiated phenotype. The damaging effects of OS and the chondroprotective activity of SPD were assessed by evaluating the extent of cell death, oxidative DNA damage and caspase 3 activation. The autophagy promoting activity of SPD was evaluated by assessing pivotal autophagic effectors, i.e. Beclin-1 (BECN-1), microtubule-associated protein 1 light chain 3 II (LC3-II) and p62. BECN-1 protein expression was significantly increased by SPD and reduced by H2O2 treatment. SPD also rescued the impaired autophagic flux consequent to H2O2 exposure by increasing mRNA and protein expression of LC3-II and p62. SPD induction of mitophagy was revealed by immunofluorescent co-localization of LC3-II and TOM20. The key protective role of autophagy was confirmed by the loss of SPD chondroprotection upon autophagy-related gene 5 (ATG5) silencing. Significant SPD tuning of the H2O2-dependent induction of degradative (MMP-13), inflammatory (iNOS, COX-2) and hypertrophy markers (RUNX2 and VEGF) was revealed by Real Time PCR and pointed at the SPD ability of reducing NF-κB activation through autophagy induction. Conversely, blockage of autophagy led to parallel increases of oxidative markers and p65 nuclear translocation. SPD also increased the proliferation of slow-proliferating primary cultures. Taken together, our findings highlight the chondroprotective, anti-oxidant and anti-inflammatory activity of SPD and suggest that the protection afforded by SPD against OS is exerted through the rescue of the autophagic flux.

Osteoarthritis (OA) is a disease associated to age or conditions that precipitate aging of articular cartilage, a post-mitotic tissue that remains functional until the failure of major homeostatic mechanisms. OA severely impacts the national health system costs and patients’ quality of life because of pain and disability. It is a whole-joint disease sustained by inflammatory and oxidative signaling pathways and marked epigenetic changes responsible for catabolism of the cartilage extracellular matrix. OA usually progresses until its severity requires joint arthroplasty. To delay this progression and to improve symptoms, a wide range of naturally derived compounds have been proposed and are summarized in this review. Preclinical in vitro and in vivo studies have provided proof of principle that many of these nutraceuticals are able to exert pleiotropic and synergistic effects and effectively counteract OA pathogenesis by exerting both anti-inflammatory and antioxidant activities and by tuning major OA-related signaling pathways. The latter are the basis for the nutrigenomic role played by some of these compounds, given the marked changes in the transcriptome, miRNome, and methylome. Ongoing and future clinical trials will hopefully confirm the disease-modifying ability of these bioactive molecules in OA patients.

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The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1

The therapeutic ability of Mesenchymal Stem/Stromal Cells to address osteoarthritis (OA) is mainly related to the secretion of biologically active factors, which can be found within their secreted Extracellular Vesicles including small Extracellular Vesicles (sEV). Aim of this study was to investigate the effects of sEV from adipose derived stromal cells (ADSC) on both chondrocytes and synoviocytes, in order to gain insights into the mechanisms modulating the inflammatory/catabolic OA environment. sEV, obtained by a combined precipitation and size exclusion chromatography method, were quantified and characterized, and administered to chondrocytes and synoviocytes stimulated with IL-1β. Cellular uptake of sEV was evaluated from 1 to 12 h. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h, when p65 nuclear translocation was investigated to study NF-κB pathway. This study underlined the potential of ADSC derived sEV to affect gene expression and protein release of both chondrocytes and synoviocytes, counteracting IL-1β induced inflammatory effects, and provided insights into their mechanisms of action. sEV uptake was faster in synoviocytes, where it also elicited stronger effects, especially in terms of cytokine and chemokine modulation. The inflammatory/catabolic environment mediated by NF-κB pathway was significantly attenuated by sEV, which hold promise as new therapeutic strategy to address OA.

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

Osteoarthritis (OA), the most prevalent degenerative joint disease, still lacks a true disease-modifying therapy. The involvement of the NF-κB pathway and its upstream activating kinases in OA pathogenesis has been recognized for many years. The ability of the N-acetyl phenylalanine glucosamine derivative (NAPA) to increase anabolism and reduce catabolism via inhibition of IKKα kinase has been previously observed in vitro and in vivo. The present study aims to confirm the chondroprotective effects of NAPA in an in vitro model of joint OA established with primary cells, respecting both the crosstalk between chondrocytes and synoviocytes and their phenotypes. This model satisfactorily reproduces some features of the previously investigated DMM model, such as the prominent induction of ADAMTS-5 upon inflammatory stimulation. Both gene and protein expression analysis indicated the ability of NAPA to counteract key cartilage catabolic enzymes (ADAMTS-5) and effectors (MCP-1). Molecular analysis showed the ability of NAPA to reduce IKKα nuclear translocation and H3Ser10 phosphorylation, thus inhibiting IKKα transactivation of NF-κB signalling, a pivotal step in the NF-κB-dependent gene expression of some of its targets. In conclusion, our data confirm that NAPA could truly act as a disease-modifying drug in OA.

Background Hydroxytyrosol (HT), a major phenolic antioxidant found in olive oil, can afford protection from oxidative stress in several types of non-tumoral cells, including chondrocytes. Autophagy was recently identified as a protective process during osteoarthritis (OA) development and critical for survival of chondrocytes. Therefore we have investigated the possibility to modulate chondrocyte autophagy by HT treatment. Methods DNA damage and cell death were estimated in human C-28/I2 and primary OA chondrocytes exposed to hydrogen peroxide. Autophagic flux and mitophagy were monitored by measuring levels and location of autophagy markers through western blot, immunostaining and confocal laser microscopy. Late autophagic vacuoles were stained with monodansylcadaverine. The involvement of sirtuin 1 (SIRT-1) was evaluated by immunohistochemistry, western blot and gene silencing with specific siRNA. Results HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires the deacetylase SIRT-1, which accumulated in the nucleus following HT treatment. In fact silencing of this enzyme prevented HT from promoting the autophagic process and cell survival. Furthermore HT supports autophagy even in a SIRT-1-independent manner, by increasing p62 transcription, required for autophagic degradation of polyubiquitin-containing bodies. Conclusions These results support the potential of HT as a chondroprotective nutraceutical compound against OA, not merely for its antioxidant ability, but as an autophagy and SIRT-1 inducer as well. General significance HT may exert a cytoprotective action by promoting autophagy in cell types that may be damaged in degenerative diseases by oxidative and other stress stimuli.

Mesenchymal stem/stromal cells (MSCs) are a reservoir for tissue homeostasis and repair that age during organismal aging. Beside the fundamental in vivo role of MSCs, they have also emerged in the last years as extremely promising therapeutic agents for a wide variety of clinical conditions. MSC use frequently requires in vitro expansion, thus exposing cells to replicative senescence. Aging of MSCs (both in vivo and in vitro) can affect not only their replicative potential, but also their properties, like immunomodulation and secretory profile, thus possibly compromising their therapeutic effect. It is therefore of critical importance to unveil the underlying mechanisms of MSC senescence and to define shared methods to assess MSC aging status. The present review will focus on current scientific knowledge about MSC aging mechanisms, control and effects, including possible anti-aging treatments.

The first step in skeleton development is the condensation of mesenchymal precursors followed by any of two different types of ossification, depending on the type of bone segment: in intramembranous ossification, the bone is deposed directly in the mesenchymal anlagen, whereas in endochondral ossification, the bone is deposed onto a template of cartilage that is subsequently substituted by bone. Polyamines and polyamine-related enzymes have been implicated in bone development as global regulators of the transcriptional and translational activity of stem cells and pivotal transcription factors. Therefore, it is tempting to investigate their use as a tool to improve regenerative medicine strategies in orthopedics. Growing evidence in vitro suggests a role for polyamines in enhancing differentiation in both adult stem cells and differentiated chondrocytes. Adipose-derived stem cells have recently proved to be a convenient alternative to bone marrow stromal cells, due to their easy accessibility and the high frequency of stem cell precursors per volume unit. State-of-the-art "prolotherapy" approaches for skeleton regeneration include the use of adipose-derived stem cells and platelet concentrates, such as platelet-rich plasma (PRP). Besides several growth factors, PRP also contains polyamines in the micromolar range, which may also exert an anti-apoptotic effect, thus helping to explain the efficacy of PRP in enhancing osteogenesis in vitro and in vivo. On the other hand, spermidine and spermine are both able to enhance hypertrophy and terminal differentiation of chondrocytes and therefore appear to be inducers of endochondral ossification. Finally, the peculiar activity of spermidine as an inducer of autophagy suggests the possibility of exploiting its use to enhance this cytoprotective mechanism to counteract the degenerative changes underlying either the aging or degenerative diseases that affect bone or cartilage.

Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3β inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore “late” effects on terminal differentiation or “early” mitochondrial effects, respectively. GSK3β inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and β-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3β inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3β enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

Osteoarthritis (OA) is a debilitating degenerative disease of the articular cartilage with a multifactorial etiology. Aging, the main risk factor for OA development, is associated with a systemic oxidative and inflammatory phenotype. Autophagy is a central housekeeping system that plays an antiaging role by supporting the clearance of senescence-associated alterations of macromolecules and organelles. Autophagy deficiency has been related to OA pathogenesis because of the accumulation of cellular defects in chondrocytes. Microribonucleic acids (microRNAs or miRs) are a well-established class of posttranscriptional modulators belonging to the family of noncoding RNAs that have been identified as key players in the regulation of cellular processes, such as autophagy, by targeting their own cognate mRNAs. Here, we present a state-of-the-art literature review on the role of miRs and autophagy in the scenario of OA pathogenesis. In addition, a comprehensive survey has been performed on the functional connections of the miR network and the autophagy pathway in OA by using “microRNA,” “autophagy,” and “osteoarthritis” as key words. Discussion of available evidence sheds light on some aspects that need further investigation in order to reach a more comprehensive view of the potential of this topic in OA.

Summary Objective Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). Methods Human primary and C-28/I2 chondrocytes were pre-treated with 100 μM HT 30 min before 100 μM H 2 O 2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. Results MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H 2 O 2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H 2 O 2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. Conclusions These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

Summary Objective Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro . The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). Design 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. Results The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. Conclusions NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo .

In the context of OA disease, NF-kappaB transcription factors can be triggered by a host of stress-related stimuli including pro-inflammatory cytokines, excessive mechanical stress and ECM degradation products. Activated NF-kappaB regulates the expression of many cytokines and chemokines, adhesion molecules, inflammatory mediators, and several matrix degrading enzymes. NF-kappaB also influences the regulated accumulation and remodeling of ECM proteins and has indirect positive effects on downstream regulators of terminal chondrocyte differentiation (including beta-catenin and Runx2). Although driven partly by pro-inflammatory and stress-related factors, OA pathogenesis also involves a "loss of maturational arrest" that inappropriately pushes chondrocytes towards a more differentiated, hypertrophic-like state. Growing evidence points to NF-kappaB signaling as not only playing a central role in the pro-inflammatory stress-related responses of chondrocytes to extra- and intra-cellular insults, but also in the control of their differentiation program. Thus unlike other signaling pathways the NF-kappaB activating kinases are potential therapeutic OA targets for multiple reasons. Targeted strategies to prevent unwanted NF-kappaB activation in this context, which do not cause side effects on other proteins or signaling pathways, need to be focused on the use of highly specific drug modalities, siRNAs or other biological inhibitors that are targeted to the activating NF-kappaB kinases IKKalpha or IKKbeta or specific activating canonical NF-kappaB subunits. However, work remains in its infancy to evaluate the effects of efficacious, targeted NF-kappaB inhibitors in animal models of OA disease in vivo and to also target these strategies only to affected cartilage and joints to avoid other undesirable systemic effects.

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N1,N11-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-α (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis. J. Cell. Physiol. 216: 153–161, 2008. © 2008 Wiley-Liss, Inc.

Objective Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKα and IKKβ are essential kinases that activate NF-κB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy. Methods Expression of IKKα or IKKβ was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed. Results The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKα or IKKβ. Silencing of IKKβ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKα dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKα-deficient cells retained the phenotype of cells in a pre–hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses. Conclusion IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKα were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKα activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

Background: The non-canonical NF-kappa B activating kinase IKK alpha, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti-inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKK alpha as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKK alpha expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKK alpha(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKK alpha in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKK alpha ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKK alpha-deficient chondrocytes. IKK alpha deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of. Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKK alpha-deficient chondrocytes was rescued by a kinase-dead IKK alpha protein mutant. Conclusions/Significance: IKK alpha acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKK alpha positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. T eta nu sigma, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

The molecular mechanisms underlying spermine osteo-inductive activity on human adipose-derived stem cells (ASCs) grown in 3-dimensional (3D) cultures were investigated. Spermine belongs to the polyamine family, naturally occurring, positively charged polycations that are able to control several cellular processes. Spermine was used at a concentration close to that found in platelet-rich plasma (PRP), an autologous blood product containing growth and differentiation factors, which has recently become popular in in vitro and in vivo bone healing and engineering. Adipose tissue was surgically obtained from the hypodermis of patients undergoing hip arthroplasty. Patient age negatively affected both ASC yield and ASC ability to form 3D constructs. ASC 3D cultures were seeded in either non differentiating or chondrogenic conditions, with or without the addition of 5 mu M spermine to evaluate its osteogenic potential across 1, 2, and 3 weeks of maturation. Osteogenic medium was used as a reference. The effects of the addition of spermine on molecular markers of osteogenesis, at both gene and protein level, and mineralization were evaluated. The effects of spermine were temporally defined and responsible for the progression from the early to the mature osteoblast differentiation phases. Spermine initially promoted gene and protein expression of Runx-2, an early marker of the osteoblast lineage; then, it increased beta-catenin expression and activation, which led to the induction of Osterix gene expression, the mature osteoblast commitment factor. The finding that spermine induces ASC to differentiate toward mature osteoblasts supports the use of these easily accessible mesenchymal stem cells coupled with PRP for orthopedic applications.

Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of environmental changes. In mammalian tissues a deficit of these mechanisms is commonly associated with cellular aging and degenerative diseases related to aging, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. Many of these proteins are enzymes that positively or negatively modulate the autophagic process. This review focuses on biochemical mechanisms involving enzymes--specifically, mTOR, AMPK, and Sirt1--that are currently considered important for these adaptive responses, providing an overview of the interactions of the main players in this process.

CXCR2 ligands contribute to chondrocyte hypertrophy and apoptosis, important determinants in cartilage pathophysiology. We unraveled the kinetics of signaling, biochemical, transcriptional, and morphological events triggered by GROα in human osteoarthritic chondrocytes kept in three-dimensional culture. p38 MAPK activation was assessed with a highly sensitive ELISA. Effector caspase activation was evaluated by cleavage of a fluorogenic substrate. Gene expression of key markers of hypertrophy (MMP-13, Runx-2) and matrix synthesis (aggrecan), and of cathepsin B isoform CB(-2,3) was evaluated by real time PCR. Occurrence of the morphological markers of apoptosis was investigated by transmission electron microscopy (TEM). GROα led to p38 MAPK activation in passaged chondrocytes cultured in micromass but not as a high-density monolayer. This caused the downstream triggering of chondrocyte hypertrophy (MMP-13 and Runx-2 upregulation, and calcium deposition) and apoptosis/anoikis following concurrence of matrix degrading activity, and inhibition of matrix synthesis which also involved the induction of CB(-2,3). These phenomena proved to be dependent on the co-receptor role of sulfated glycosaminoglycans (sGAG) and the activation of p38 MAPK, since they were abrogated either by preincubation with soluble chondroitin-4 sulfate or p38 MAPK inhibitors. The co-receptor role of sGAG was further demonstrated by colocalization experiments of these molecules with GROα in the stimulated micromasses. These findings suggest that extracellular matrix exerts a regulatory role in chondrocytes differentiation, and that meaningful investigation of the effects of chemokines on chondrocyte biology requires culture conditions respectful of both the differentiated status of the chondrocytes and of their three-dimensional interaction with the extracellular matrix. J. Cell. Physiol. 210: 417–427, 2007. © 2006 Wiley-Liss, Inc.

Chondrocyte apoptosis can be an important contributor to cartilage degeneration, thereby making it a potential therapeutic target in articular diseases. To search for new approaches to limit chondrocytic cell death, we investigated the requirement of polyamines for apoptosis favored by tumor necrosis factor-alpha (TNF), using specific polyamine biosynthesis inhibitors in human chondrocytes. The combined treatment of C-28/I2 chondrocytes with TNF and cycloheximide (CHX) resulted in a prompt effector caspase activation and internucleosomal DNA fragmentation. Pre-treatment of chondrocytes with alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, markedly reduced putrescine and spermidine content as well as the caspase-3 activation and DNA fragmentation induced by TNF and CHX. DFMO treatment also inhibited the increase in effector caspase activity provoked by TNF plus MG132, a proteasome inhibitor. DFMO decreased caspase-8 activity and procaspase-8 content, an apical caspase essential for TNF-induced apoptosis. Although DFMO increased the amount of active, phosphorylated Akt, inhibitors of the Akt pathway failed to restore the TNF-induced increase in caspase activity blunted by DFMO. DFMO also reduced the increase in caspase activity induced by staurosporine, but in this case Akt inhibition prevented the DFMO effect. Pre-treatment with CGP 48664, an S-adenosylmethionine decarboxylase (SAMDC) inhibitor markedly reduced spermidine and spermine levels, and provoked effects similar to those caused by DFMO. Finally DFMO was effective even in primary osteoarthritis (OA) chondrocyte cultures. These results suggest that the intracellular depletion of polyamines in chondrocytes can inhibit both the death receptor pathway by reducing the level of procaspase-8, and the apoptotic mitochondrial pathway by activating Akt.

Besides the well-known activities of the prototypical inflammatory cytokines (IL-1beta, TNFalpha), a role for chemokines and their receptors in cartilage degradation in osteoarthritis has recently been reported. Human chondrocytes can produce CC and CXC chemokines and express chemokine receptors for both chemokine subfamilies. Engagement of these receptors can induce the release of matrix degrading enzymes such as matrix metalloproteinases 1, 3, and 13, and N-acetyl-beta-D-glucosaminidase. Furthermore GROalpha, a CXC chemokine acting on CXCR2, can activate an apoptotic pathway in chondrocytes that leads to chondrocyte cell death. These findings suggest that chemokines can act as an autocrine or paracrine loop on chondrocytes and can contribute to many pathophysiological patterns present in osteoarthritis. Chemokines and their downstream signaling pathways can be considered novel therapeutic targets in osteoarthritis.

Objective. To evaluate in vivo expression of chemokine receptors in cartilage tissue samples from healthy and diseased joints. Methods. Presence and distribution of several chemokine receptors in cartilage samples from patients with osteoarthritis (OA) or inflammatory arthritis (IA) and from multi-organ donors were assessed by immunohistochemistry. The expression of messenger RNA (mRNA) for chemokine receptors was also analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Normal and OA-affected cartilage showed a moderate to high expression of chemokine receptors, while staining of IA samples ranged from low to absent. Differences between OA and IA samples were present for all receptors but CCR2 and CXCR4. Moreover, mRNAs for CCR1, CCR5 and CXCR1 were found both in normal and pathological chondrocytes, suggesting that chemokine receptor down-modulation seen in IA samples could be a post-transcriptional event. Conclusion. Data on normal and pathological chondrocytes underline the role of chemokines in cartilage homeostasis and suggest an imbalance towards catabolic processes in inflammatory conditions.

Background In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. Methodology/Principal Findings The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. Conclusions/Significance Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

Chemokines play a key role in modulating leukocyte functions at sites of inflammation. To assess chondrocyte contribution to the chemotactic environment of inflamed joints the intracellular content of CC and CXC chemokines was investigated. IL-8, GROalpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression was evaluated by flow cytometric analysis and RT-PCR in chondrocytes isolated from cartilage specimens obtained from patients with osteoarthritis and rheumatoid arthritis and multiorgan donors as normal controls. All the chemokines except RANTES were found in normal chondrocytes, with different degrees of staining intensity. In osteoarthritis and rheumatoid arthritis patients, an enhancement of IL-8, GROalpha, MIP-1alpha and MIP-1beta was observed.

In HIV+ patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-1. The cells were fixed in suspension and hybridized by means of an HIV-1 genomic probe labeled with digoxigenin-11-dUTP. An FITC-labeled anti-digoxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Flow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples.

Different immunological techniques were compared for their sensitivity in detecting some important autoantibodies in the sera of patients with rheumatic diseases. Sera of patients with systemic lupus erythematosus were screened for anti-dsDNA, Sm, and RNP autoantibodies by Crithidia luciliae assay, Farr assay, enzyme immunoassay, and immunoblotting. Sera of patients with Sjögren's syndrome were screened for anti-SSA and anti-SSB antibodies by enzyme immunoassay, counter immunoelectrophoresis, and immunoblotting and sera of patients with scleroderma for SCL-70 autoantibodies by enzyme immunoassay counter immunoelectrophoresis, and immunofluorescence on Hep-2 cells. Enzyme immunoassay and counter immunoelectrophoresis gave the most positive results and the best agreement compared with the other techniques. Immunofluorescence gave few positive results for each antibody evaluated. Immunoblotting gave intermediate results for all autoantibodies except anti-SSA, where the prevalence was low. There was no relationship between levels of dsDNA, Sm, and RNP antibodies and disease activity score in systemic lupus erythematosus patients.