Maintaining Cartilage Homeostasis Research Articles

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

Suppression of NF-κB activity via nanoparticle-based siRNA delivery alters early cartilage responses to injury

Osteoarthritis (OA) is a major cause of disability and morbidity in the aging population. Joint injury leads to cartilage damage, a known determinant for subsequent development of posttraumatic OA, which accounts for 12% of all OA. Understanding the early molecular and cellular responses postinjury may provide targets for therapeutic interventions that limit articular degeneration. Using a murine model of controlled knee joint impact injury that allows the examination of cartilage responses to injury at specific time points, we show that intraarticular delivery of a peptidic nanoparticle complexed to NF-κB siRNA significantly reduces early chondrocyte apoptosis and reactive synovitis. Our data suggest that NF-κB siRNA nanotherapy maintains cartilage homeostasis by enhancing AMPK signaling while suppressing mTORC1 and Wnt/β-catenin activity. These findings delineate an extensive crosstalk between NF-κB and signaling pathways that govern cartilage responses postinjury and suggest that delivery of NF-κB siRNA nanotherapy to attenuate early inflammation may limit the chronic consequences of joint injury. Therapeutic benefits of siRNA nanotherapy may also apply to primary OA in which NF-κB activation mediates chondrocyte catabolic responses. Additionally, a critical barrier to the successful development of OA treatment includes ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Here, we show that the peptide-siRNA nanocomplexes are nonimmunogenic, are freely and deeply penetrant to human OA cartilage, and persist in chondrocyte lacunae for at least 2 wk. The peptide-siRNA platform thus provides a clinically relevant and promising approach to overcoming the obstacles of drug delivery to the highly inaccessible chondrocytes.

Inflammatory conditions partly impair the mechanically mediated activation of Smad2/3 signaling in articular cartilage.

BackgroundJoint trauma, which is frequently related with mechanical overloading of articular cartilage, is a well-established risk for osteoarthritis (OA) development. Additionally, reports show that trauma leads to synovial joint inflammation. In consequence, after joint trauma, cartilage is influenced by deleterious excessive loading combined with the catabolic activity of proinflammatory mediators. Since the activation of TGF-β signaling by loading is considered to be a key regulatory pathway for maintaining cartilage homeostasis, we tested the effect of proinflammatory conditions on mechanically mediated activation of TGF-β/Smad2/3P signaling in cartilage.MethodsCartilage explants were subjected to dynamic mechanical compression in the presence of interleukin-1 beta (IL-1β) or osteoarthritic synovium-conditioned medium (OAS-CM). Subsequently, the activation of the Smad2/3P pathway was monitored with QPCR analysis of reporter genes and additionally the expression of receptors activating the Smad2/3P pathway was analyzed. Finally, the ability for mechanically mediated activation of Smad2/3P was tested in human OA cartilage.ResultsIL-1β presence during compression did not impair the upregulation of Smad2/3P reporter genes, however the results were affected by IL-1β-mediated upregulations in unloaded controls. OAS-CM significantly impaired the compression-mediated upregulation of bSmad7 and Tgbfb1. IL-1β suppressed the compression-mediated bAlk5 upregulation where 12 MPa compression applied in the presence of OAS-CM downregulated the bTgfbr2. Mechanically driven upregulation of Smad2/3P reporter genes was present in OA cartilage.ConclusionsProinflammatory conditions partly impair the mechanically mediated activation of the protective TGF-β/Smad2/3P pathway. Additionally, the excessive mechanical compression, applied in the presence of proinflammatory conditions diminishes the expression of the type II TGF-β receptor, a receptor critical for maintenance of articular cartilage.

Chondromodulin-1 ameliorates osteoarthritis progression by inhibiting HIF-2α activity

Hypoxia is known to stabilize hypoxia-inducible factor (HIF) and initiate angiogenic signaling cascade. However, cartilage living in hypoxia environment can maintain avascularity. It is well known that abrogation of avascularity is related to cartilage degradation in osteoarthritis (OA). The aims of present study were to investigate the role of chondromodulin-1 (ChM-1), an endogenously anti-angiogenic protein in cartilage, during chondrocyte maturation and OA progression, as well as to explore the molecular mechanisms underlying the function of ChM-1 with a focus on HIF-2α pathway. Angiogenic-related markers were evaluated in OA cartilage and different stages of chondrocyte differentiation. Chondrocytes transfected with ChM-1 lentivirus or siRNA was treated with tumor necrosis factor (TNF-α) to investigate the role of ChM-1 in chondrocyte hypertrophic changes. Invivo study was conducted by using a surgical induced OA rat model with intra-articular injection of lentivirus ChM-1 (LV-ChM-1) or mock lentivirus (LV-GFP) control. Transcriptional activity of HIF-2α was determined by chromatin immunoprecipitation (ChIP) assay to unveil the mechanisms of ChM-1. Majority angiogenic factors increased in severe OA cartilage, while anti-angiogenic factors including ChM-1 decreased. ChM-1 expression was strongly related with chondrocyte differentiation and chondrogenesis invitro. ChM-1 overexpression protected chondrocytes from TNF-α induced hypertrophy, and intra-articular injection of LV-ChM-1 delayed OA progression. ChM-1 delayed HIF-2α nuclear translocation at early time-points and decreased transcriptional activity of HIF-2α on collagen type Х α1 (COL10A1), vascular endothelial growth factor A (VEGFA) and matrix metallopeptidase-13 (MMP-13). ChM-1 maintains cartilage homeostasis by inhibiting HIF-2α induced catabolic activity and regulation of ChM-1 in cartilage may be a promising therapeutic strategy for OA.

The Role of Sirtuins in Cartilage Homeostasis and Osteoarthritis.

The past decade has witnessed many advances in the understanding of sirtuin biology and related regulatory circuits supporting the capacity of these proteins to serve as energy-sensing molecules that contribute to healthspan in various tissues, including articular cartilage. Hence, there has been a significant increase in new investigations that aim to elucidate the mechanisms of sirtuin function and their roles in cartilage biology, skeletal development, and pathologies such as osteoarthritis (OA), rheumatoid arthritis (RA), and intervertebral disc degeneration (IVD). The majority of the work carried out to date has focused on SIRT1, although SIRT6 has more recently become a focus of some investigations. In vivo work with transgenic mice has shown that Sirt1 and Sirt6 are essential for maintaining cartilage homeostasis and that the use of sirtuin-activating molecules such as resveratrol may have beneficial effects on cartilage anabolism. Current thinking is that SIRT1 exerts positive effects on cartilage by encouraging chondrocyte survival, especially under stress conditions, which may provide a mechanism supporting the use of sirtuin small-molecule activators (STACS) for future therapeutic interventions in OA and other degenerative pathologies of joints, especially those that involve articular cartilage.

MiR-105/Runx2 axis mediates FGF2-induced ADAMTS expression in osteoarthritis cartilage.

Fibroblast growth factor 2 (FGF2) plays an important role in the development of osteoarthritis (OA) through the regulation of cartilage degradation. However, the molecular mechanism underlying FGF2-induced OA is poorly characterized. MicroRNAs (miRNAs) maintain cartilage homeostasis. To examine whether FGF2 regulates OA through the modulation of miRNA, we screened potential miRNA molecules that could be regulated through FGF2 using microarray analysis. The results showed that microRNA-105 (miR-105) was significantly downregulated in chondrocytes stimulated with FGF2. Runt-related transcription factor 2 (Runx2), a key transcription factor involved in OA, has been identified as a novel potential target of miR-105. FGF2 suppressed miR-105 expression through the recruitment of the subunit of the nuclear factor kappa B transcription complex p65 to the miR-105 promoter. The knockdown of Runx2 mimicked the effect of miR-105 and abolished the ability of miR-105 to regulate the expression of a disintegrin-like and metalloproteinase with thrombospondin 4 (ADAMTS4), ADAMTS5, ADAMTS7 and ADAMTS12, both of which are responsible for the degradation of collagen 2A1 (COL2A1) and aggrecan (ACAN). miR-105 is also required for FGF2/p65-induced Runx2 activation and ADAMTS expression. Moreover, miR-105 expression was downregulated in OA patients and inversely correlated with the expression of Runx2, ADAMTS7 and ADAMTS12, which were upregulated in OA patients. These data highlight that the FGF2/p65/miR-105/Runx2/ADAMTS axis might play an important role in OA pathogenesis and that miR-105 might be a potential diagnostic target and useful strategy for OA treatment. Runx2 was identified as a novel direct target of miR-105. FGF2 inhibits miR-105 transcription through recruitment of p65 to miR-105 promoter. p65/miR-105 is essential for FGF2-mediated Runx2 and ADAMTS upregulation. miR-105 is downregulated in OA and inversely correlated with Runx2 expression.

Synovial fluid concentration of gremlin1 but not frizzled-related protein is increased in patients after acute knee injury

Purpose: Degenerative joint diseases are characterized by a disturbance in joint homeostasis, which is poorly understood at the molecular level. Previously, we have identified the secreted WNT antagonists dickkopf 1 homolog (Xenopus laevis) (DKK1) and frizzled-related protein (FRZB) and the BMP antagonists Gremlin1 (GREM1) as regulators of cartilage homeostasis by preventing hypertrophic differentiation of articular chondrocytes, consistent with their role in articular cartilage homeostasis. In this study we tested the hypothesis that health status of the joint is reflected by the expression levels of FRZB and GREM1 in synovial fluid. To test this hypothesis, we performed a cross sectional analysis of synovial fluid samples collected from patients with acute and chronic knee injury and compared these with synovial fluid samples from knee healthy controls. Methods: Synovial fluid was aspirated (Lund University, Sweden) from patients with acute (n =106, mean age 30 years [range 17-57], 0-77 days from injury) and chronic (n = 50, mean age 38 years [18-70] 1-37 years after injury) knee injuries, and from knee healthy subjects (n = 8, mean age 28 years [17-85]). Synovial fluid concentrations of FRZB and GREM1 were determined using ELISA (FRZB, RD GREM1, Bio-connect diagnostics). Concentration of cartilage biomarkers in synovial fluid; ARGS-aggrecan (in-house), cartilage oligomeric matrix protein (COMP; BioVendor) and collagen epitope C2C (IBEX), and proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α; Meso Scale Discovery) were analyzed by immunoassays. Mann-Whitney tests for group comparisons and Spearman’s rank test (rS) for correlation analysis was used. Results: Synovial fluid concentration of FRZB in the acute injury group was no different from the level in the reference group (p = 0.99), but was 2-fold (based on median concentrations) higher than the level of the chronic injury group (p = 0.023) (Fig. 1a). Synovial fluid concentrations of GREM1 in the acute and chronic injury groups were both higher than the level of the reference group, 2-fold (p = 0.003) and 1.8-fold (p = 0.031), respectively (Fig. 1b); also, in these patients the GREM1 concentration was negatively correlated to time after injury (rS = -0.224, p = 0.005). The FRZB and GREM1 concentrations of the acute and chronic injury groups were not influenced by gender (p = 0.550, p = 0.825) or by age (rS =-0.146, p = 0.068; rS = -0.137, p = 0.089), although, the FRZB concentration was significantly decreased in knee healthy subjects with age (rS= -0.833, p = 0.005). In the acute and chronic injury groups, there was no correlation between FRZB and GREM1 concentrations in synovial fluid (rS = 0.04, p = 0.585). Also, for a portion of the acute injury group (n = 52) synovial fluid FRZB concentration were correlated with COMP levels (rS = 0.284, p = 0.041) but not with other cartilage markers (C2C p = 0.55, ARGS-aggrecan p = 0.311) nor with any cytokine levels (p> 0.05 for all cytokines); the synovial fluid GREM1 concentration did not correlated with any cartilage marker nor with cytokine levels (p > 0.05 for all markers). Conclusions: Compared to chronic injured knees, acute injured knees have higher synovial fluid concentrations of FRZB and GREM1. Their levels were not influenced by gender or age, indicating that the differences of FRZB and GREM1 concentrations between acute and chronic injured knee joints depend on differences in disease conditions. Persistent synovial fluid levels of FRZB in acute injured knee joints might be a way for cells trying to protect the cartilage from degradation by inhibiting the Wnt signaling pathway. Similarly, high synovial fluid levels of GREM1 in injured knee joints (acute and chronic) suggests of an effort to maintain cartilage homeostasis by inhibiting BMP signaling.

O-Phenanthroline as modulator of the hypoxic and catabolic response in cartilage tissue-engineering models.

Hypoxia has been shown to be important for maintaining cartilage homeostasis as well as for inducing chondrogenic differentiation. Ensuring low oxygen levels during in vitro culture is difficult, therefore we assessed the chondro-inductive capabilities of the hypoxia-mimicking agent O-phenanthroline, which is also known as a non-specific matrix metalloproteinase (MMP) inhibitor. We found that O-phenanthroline reduced the expression of MMP3 and MMP13 mRNA levels during chondrogenic differentiation of human chondrocytes (hChs), as well as after TNFα/IL-1β exposure in an explant model. Interestingly, O-phenanthroline significantly inhibited matrix degradation in a TNFα/IL-1β-dependent model of cartilage degeneration when compared to control and natural hypoxia (2.5% O2 ). O-Phenanthroline had limited ability to improve the chondrogenic differentiation or matrix deposition in the chondrogenic pellet model. Additionally, O-phenanthroline alleviated MMP-induced cartilage degradation without affecting chondrogenesis in the explant culture. The data presented in this study indicate that the inhibitory effect of O-phenanthroline on MMP expression is dominant over the hypoxia-mimicking effect. Copyright © 2014 John Wiley & Sons, Ltd.

The critical role of UDP-galactose-4-epimerase in osteoarthritis: Modulating proteoglycans synthesis of the articular chondrocytes

UDP-galactose-4-epimerase (GALE) is a key enzyme catalyzing the interconversion of UDP-glucose and UDP-galactose, as well as UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which are all precursors for the proteoglycans (PGs) synthesis. However, whether GALE is essential in cartilage homeostasis remains unknown. Therefore, we investigated the role of GALE in PGs synthesis of human articular chondrocytes, the GALE expression in OA, and the regulation of GALE expression by interleukin-1beta (IL-1β). Silencing GALE gene with specific siRNAs resulted in a markedly inhibition of PGs synthesis in human articular chondrocytes. GALE protein levels were also decreased in both human and rat OA cartilage, thus leading to losses of PGs contents. Moreover, GALE mRNA expression was stimulated by IL-1β in early phase, but suppressed in late phase, while the suppression of GALE expression induced by IL-1β was mainly mediated by stress-activated protein kinase/c-Jun N-terminal kinase pathway. These data indicated a critical role of GALE in maintaining cartilage homeostasis, and suggested that GALE inhibition might contribute to OA progress.

A homeostatic function of CXCR2 signalling in articular cartilage

ObjectiveELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells....

The anti-catabolic role of bovine lactoferricin in cartilage.

Bovine lactoferricin (LfcinB) is a multifunctional peptide derived from bovine lactoferrin that demonstrates antibacterial, antifungal, antiviral, antitumor, and immunomodulatory activities. Recently, studies have focused on the anti-catabolic and anti-inflammatory potential of LfcinB. LfcinB is able to modulate the effects cytokines such as IL-1 and fibroblast growth factor 2 as well as promote specific cartilage anabolic factors. These properties are particularly important in maintaining cartilage homeostasis and preventing a catabolic state, which leads to clinical pathology. This review focuses on the recent literature elucidating the role of LfcinB in preventing cartilage degradation.

Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells

Physiologic mechanical stress stimulates expression of chondrogenic genes, such as multifunctional growth factor CYR61/CTGF/NOV (CCN) 2 and α1(II) collagen (COL2A1), and maintains cartilage homeostasis. In our previous studies, cyclic tensile strain (CTS) induces nuclear translocation of transforming growth factor (TGF)-β receptor-regulated Smad2/3 and the master chondrogenic transcription factor Sry-type HMG box (SOX) 9. However, the precise mechanism of stretch-mediated Smad activation remains unclear in transcriptional regulation of CCN2 and COL2A1. Here we hypothesized that CTS may induce TGF-β1 release and stimulate Smad-dependent chondrogenic gene expression in human chondrocytic SW1353 cells. Uni-axial CTS (0.5Hz, 5% strain) stimulated gene expression of CCN2 and COL2A1 in SW1353 cells, and induced TGF-β1 secretion. CCN2 synthesis and nuclear translocalization of Smad2/3 and SOX9 were stimulated by CTS. In addition, CTS increased the complex formation between phosphorylated Smad2/3 and SOX9. The CCN2 promoter activity was cooperatively enhanced by CTS and Smad3 in luciferase reporter assay. Chromatin immunoprecipitation revealed that CTS increased Smad2/3 interaction with the CCN2 promoter and the COL2A1 enhancer. Our results suggest that CTS epigenetically stimulates CCN2 transcription via TGF-β1 release associated with Smad2/3 activation and enhances COL2A1 expression through the complex formation between SOX9 and Smad2/3.

Ibalance osteotomy in early treatment of osteoarthritis

Ibalance osteotomy in early treatment of osteoarthritis

Role of insulin‐like growth factors (IGFs), their receptors and genetic regulation in the chondrogenesis and growth of the mandibular condylar cartilage

Growth of the mandibular condylar cartilage (MCC) is reviewed as a function of genetic and epigenetic factors. The growth centers around the differential spatial concentration of the chondrocytes, influence of growth factors like TGF-β and heterogeneity in the number of IGF receptors, control the action of IGF. Besides these factors, growth of the mandibular condyle is influenced by differential response of chondrocytes as a function of their source/ageing, which in turn is regulated by TGF-β, BMPs and IGFs. While IGF-1 promotes proteoglycan synthesis and survival of the chondrocytes to maintain cartilage homeostasis, TGF-β synergistically catalysed the effect of IGF-1, while BMPs catalysed proteolysis as and when physiologically needed. To understand these processes, role of IGF-1 and its six receptors is at the center to a number of physiological processes being regulated by its mode of application for the growth and differentiation. Probing deeper, biological functions of IGFs seemed to depend on their level of free status rather than bound status to respective IGF-binding proteins (IGF-BPs), considered prerequisite to modulate their biological functions. Genetic regulation of their secretion has thrown light on their insulin-like structural homology, level and response in osteo-arthritis (OA), rheumatic arthritis (RA) and diabetes type-II. Biochemistry and spatial distribution of IGF receptors in different domains exerts control on IGF-1 activities. In ultimate analysis, IGF-axis conserved during the evolution to regulate cell growth and proliferation affect nearly every organ in the body as judged from the techniques determining skeletal maturity and decision making dependent on it for orthodontic, orthognathic/orthopedic and dental implant applications.

Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1(-/-) and Sulf-2(-/-) mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf(-/-) mice. Articular cartilage and cultured chondrocytes from Sulf(-/-) mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm 2 for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2α mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2α is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

New insights into osteoarthritis: early developmental features of an ageing-related disease

This review addresses possible common mechanisms of how recently identified consistent osteoarthritis susceptibility genes influence both the onset of osteoarthritis and its progression towards clinical outcomes. Genetic association studies have identified a few consistent osteoarthritis susceptibility genes (FRZB, GDF5, and DIO2) that replicate across different populations. Remarkably, each of these genes appears to be primarily involved in the endochondral ossification processes. We hypothesize that these osteoarthritis susceptibility genes may play a dual negative role. In early developmental processes, they may involve aberrant skeletal morphogenesis leading to either malformation of joints or aberrant bone composition or both, thereby increasing the biomechanical burden on the articular cartilage surface. Later in life in articular cartilage, these genes may affect the propensity of articular chondrocytes to become hypertrophic. As hypertrophic chondrocytes are not able to maintain cartilage homeostasis, these genes may, in part, be responsible for both the onset of osteoarthritis and the progression towards clinical outcomes. Major therapeutic advances may come from a focus on factors that enhance phenotypic stability of the articular chondrocyte during life, promoting the healthy articular cartilage.

Distinguishing Aggrecan Loss from Aggrecan Proteolysis in ADAMTS-4 and ADAMTS-5 Single and Double Deficient Mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.

The role of HIF-1α in maintaining cartilage homeostasis and during the pathogenesis of osteoarthritis

As a consequence of the avascular nature of cartilage the microenvironment in which chondrocytes must exist is characterized by hostile conditions, most prominently very low levels of oxygen (hypoxia). In recent years, a vast number of papers reporting on the role of hypoxia in cartilage development and disease has been published. It is well established today that the principal mediator of cellular adaptation to hypoxia, the transcription factor hypoxia inducible factor (HIF)-1, is of pivotal importance for survival and growth arrest of chondrocytes during cartilage development as well as energy generation and matrix synthesis of chondrocytes in healthy as well as osteoarthritic cartilage. With this commentary we aim to briefly discuss the recently published literature in this field.

Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan–CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte–hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bovine articular chondrocytes, treatment with hyaluronan oligosaccharides or purified hyaluronan hexasaccharides induced the production of nitric oxide that mirrored nitric oxide production following interleukin-1 treatment. In contrast, 120 and 1260 kDa hyaluronan did not induce production of nitric oxide. Human chondrocytes responded similarly to treatment with hyaluronan or hyaluronan oligosaccharides. Nitric oxide production from chondrocytes was mediated by activation of the inducible nitric oxide synthase, as confirmed by mRNA expression and inhibition of nitric oxide production by diphenyleneiodonium. Co-treatment of chondrocytes with hyaluronan oligosaccharides and interleukin-1 did not demonstrate additive effects. Blocking interleukin-1 receptors with an antagonist did not abolish the production of nitric oxide induced by treatment with hyaluronan oligosaccharides. Moreover, only COS-7 following transfection with a pCD44, not the CD44-null parental cells, responded to treatment with hyaluronan oligosaccharides by releasing nitric oxide. This study demonstrates a novel signaling potential by hyaluronan fragments, in lieu of endogenous hyaluronan–chondrocyte interactions, resulting in the activation of inducible nitric oxide synthase.