Midkine promotes articular chondrocyte proliferation through the MK-LRP1-nucleolin signaling pathway

Osteoarthritis (OA) is the most common disease of joint tissues; unfortunately, there are currently no curative therapies available for OA. Chondrocytes, the only cell type residing in cartilage, secrete many types of collagen (the mainly one is type II collagen) and aggrecan, which are the main components of the cartilage matrix. Chondrocyte apoptosis can lead to OA degenerative progression. We previously indicated that recombinant human midkine (rhMK), as a chondrocyte growth factor has a significant reparative effect on cartilage injury animal models. However, the molecular mechanism of this restorative function remains under investigation. Herein, we focused on the molecular mechanism underlying the role of MK in promoting the proliferation of chondrocytes cultured in vitro. Chondrocytes from rats and OA patients were successfully isolated by the digestion of articular cartilage using type II collagenase, and their proliferation was evaluated by a CCK8 assay and flow cytometry. rhMK stimulated the proliferation of chondrocytes from both OA patients and rats. Furthermore, qRT-PCR, shRNA-mediated knockdown, Western blot and immunoprecipitation (IP) assays were performed to identify the receptor and key elements responsible for the role of MK in promoting chondrocyte proliferation. Low-density lipoprotein receptor-related protein 1 (LRP1) was identified as the dominant MK receptor in chondrocytes that, as a translocator, mediates the endocytosis of MK. After being transferred into chondrocytes, MK was shown to form a complex with nucleolin that interacts with the active form of K-Ras. Upon the activation of ERK1/2, cyclin D1 expression was upregulated, promoting the chondrocyte cell cycle. Our data reveal for the first time the role of the MK-LRP1-nucleolin signaling pathway in facilitating MK-induced chondrocyte proliferation, thus providing a strong theoretical foundation for the further use of MK in OA clinical therapy.     

Therapeutic effects of bone marrow mesenchymal stem cells-derived exosomes on osteoarthritis

Mesenchymal stem cells (MSCs) have shown chondroprotective effects in clinical models of osteoarthritis (OA). However, effects of MSC-derived exosomes on OA remain unclear. The study aimed to investigate the therapeutic potential of exosomes from human bone marrow MSCs (BM-MSCs) in alleviating OA. The anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery were performed on the knee joints of a rat OA model, followed by intra-articular injection of BM-MSCs or their exosomes. In addition, BM-MSC-derived exosomes were administrated to primary human chondrocytes to observe the functional and molecular alterations. Both of BM-MSCs and BM-MSC-derived exosomes alleviated cartilage destruction and subchondral bone remodelling in OA rat model. Administration of BM-MSCs and exosomes could reduce joint damage and restore the trabecular bone volume fraction, trabecular number and connectivity density of OA rats. In addition, in vitro assays showed that BM-MSCs-exosomes could maintain the chondrocyte phenotype by increasing collagen type II synthesis and inhibiting IL-1β–induced senescence and apoptosis. Furthermore, exosomal lncRNA MEG-3 also reduced the senescence and apoptosis of chondrocytes induced by IL-1β, indicating that lncRNA MEG-3 might partially account the anti-OA effects of BM-MSC exosomes. The exosomes from BM-MSCs exerted beneficial therapeutic effects on OA by reducing the senescence and apoptosis of chondrocytes, suggesting that MSC-derived exosomes might provide a candidate therapy for OA treatment.     

Surfactant protein D attenuates nitric oxide-stimulated apoptosis in rat chondrocyte by suppressing p38 MAPK signaling

Innate immune molecule surfactant protein D (SP-D), a member of the C-type lectin protein family, plays an indispensable role in host defense and the regulation of inflammation in the lung and other tissues. Osteoarthritis (OA) is a degenerative disease of cartilage, with inflammation that causes pathologic changes and tissue damage. However, it is unknown whether there exist SP-D expression and its potential role in the pathogenesis of OA. In this study, we examined SP-D expression and explored its biological function in a sodium nitroprusside (SNP)-stimulated rat chondrocytes and surgically-induced rat OA model. We found SP-D expression in both human and rat articular chondrocytes, with higher level in normal chondrocytes compared to in OA chondrocytes. Furthermore, In vivo study demonstrated that recombinant human SP-D (rhSP-D) ameliorated cartilage degeneration in surgically-induced rat OA model. In vitro cell culture study showed that rhSP-D markedly inhibited the expression of caspase-3 as an apoptosis biomarker, and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which resulted in maintaining normal nuclear morphology and increasing mitochondrial membrane potential in SNP-stimulated rat chondrocytes. Collectively, these findings indicate that SP-D expresses in articular chondrocytes and suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via suppressing p38 MAPK activity.     

MiR‐29b‐3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.     

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.     

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.     

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL‐1β/NF‐κB pathway

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.     

Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation

Articular cartilage is optimised for bearing mechanical loads. Chondrocytes are the only cells present in mature cartilage and are responsible for the synthesis and integrity of the extracellular matrix. Appropriate joint loads stimulate chondrocytes to maintain healthy cartilage with a concrete protein composition according to loading demands. In contrast, inappropriate loads alter the composition of cartilage, leading to osteoarthritis (OA). Matrix metalloproteinases (MMPs) are involved in degradation of cartilage matrix components and have been implicated in OA, but their role in loading response is unclear. With this study, we aimed to elucidate the role of MMP-1 and MMP-3 in cartilage composition in response to mechanical load and to analyse the differences in aggrecan and type II collagen content in articular cartilage from maximum- and minimum-weight-bearing regions of human healthy and OA hips. In parallel, we analyse the apoptosis of chondrocytes in maximal and minimal load areas. Because human femoral heads are subjected to different loads at defined sites, both areas were obtained from the same hip and subsequently evaluated for differences in aggrecan, type II collagen, MMP-1, and MMP-3 content (enzyme-linked immunosorbent assay) and gene expression (real-time polymerase chain reaction) and for chondrocyte apoptosis (flow cytometry, bcl-2 Western blot, and mitochondrial membrane potential analysis). The results showed that the load reduced the MMP-1 and MMP-3 synthesis (p < 0.05) in healthy but not in OA cartilage. No significant differences between pressure areas were found for aggrecan and type II collagen gene expression levels. However, a trend toward significance, in the aggrecan/collagen II ratio, was found for healthy hips (p = 0.057) upon comparison of pressure areas (loaded areas > non-loaded areas). Moreover, compared with normal cartilage, OA cartilage showed a 10- to 20-fold lower ratio of aggrecan to type II collagen, suggesting that the balance between the major structural proteins is crucial to the integrity and function of the tissue. Alternatively, no differences in apoptosis levels between loading areas were found – evidence that mechanical load regulates cartilage matrix composition but does not affect chondrocyte viability. The results suggest that MMPs play a key role in regulating the balance of structural proteins of the articular cartilage matrix according to local mechanical demands.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Baicalein alleviates osteoarthritis by protecting subchondral bone,inhibiting angiogenesis and synovial proliferation

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.     

Invited review: the mitochondrion in osteoarthritis

In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage promote aging, cell death, and ultimately, functional failure and degeneration. Because articular cartilage chondroyctes are highly glycolytic, mitochondrially mediated pathogenesis has not been previously applied in models for pathogenesis of osteoarthritis (OA), a cartilage degenerative disease that increases markedly in aging. However, chondrocyte mitochondria respire in vitro and they demonstrate swelling and changes in number in situ in the course of OA. Normal chondrocyte mitochondrial function is hypothesized to critically support adenosine triphosphate (ATP) reserves in functional stressed chondrocytes during OA evolution. In this model, disruption of chondrocyte respiration by nitric oxide, a mediator markedly up-regulated in OA cartilage, is centrally involved in chondrocyte functional compromise. Furthermore, mitochondrial dysfunction can mediate several specific pathogenic pathways implicated in OA. These include oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, increased chondrocyte apoptosis, and pathologic cartilage matrix calcification. In addition, the direct, sublethal impairment of chondrocyte mitochondrial ATP synthesis in vitro decreases matrix synthesis and increases matrix calcification ('disease in a dish'). The weight of evidence reviewed herein strongly supports chondrocyte mitochondrial impairment as a mediator of the establishment and progression of OA.     

MiR‐155 promotes interleukin‐1β‐induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1‐mediated PI3K/Akt pathway

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation, in which elevated chondrocyte apoptosis and catabolic activity play an important role. MicroRNA‐155 (miR‐155) has recently been shown to regulate apoptosis and catabolic activity in some pathological circumstances, yet, whether and how miR‐155 is associated with OA pathology remain unexplored. We report here that miR‐155 level is significantly up‐regulated in human OA cartilage biopsies and also in primary chondrocytes stimulated by interleukin‐1β (IL‐1β), a pivotal pro‐catabolic factor promoting cartilage degradation. Moreover, miR‐155 inhibition attenuates and its overexpression promotes IL‐1β‐induced apoptosis and catabolic activity in chondrocytes in vitro. We also demonstrate that the PIK3R1 (p85α regulatory subunit of phosphoinositide 3‐kinase (PI3K)) is a target of miR‐155 in chondrocytes, and more importantly, PIK3R1 restoration abrogates miR‐155 effects on chondrocyte apoptosis and catabolic activity. Mechanistically, PIK3R1 positively regulates the transduction of PI3K/Akt pathway, and a specific Akt inhibitor reverses miR‐155 effects on promoting chondrocyte apoptosis and catabolic activity, phenocopying the results obtained via PIK3R1 knockdown, hence establishing that miR‐155 promotes chondrocyte apoptosis and catabolic activity through targeting PIK3R1‐mediated PI3K/Akt pathway activation. Altogether, our study discovers novel roles and mechanisms of miR‐155 in regulating chondrocyte apoptosis and catabolic activity, providing an implication for therapeutically intervening cartilage degradation and OA progression.     

Mitochondrial dysfunction in osteoarthritis

In osteoarthritis (OA) a time or age dependent process leads to aberrant cartilage structure which is characterized by reduced number of chondrocytes, loss of existing cartilage extracellular matrix, the production of matrix with abnormal composition and pathologic matrix calcification. Because chondrocyte matrix synthesis and mineralization are modulated by the balance between ATP generation and consumption, the mechanism by which chondrocytes generate energy have been a topic of interest. The analysis of mitochondrial respiratory chain (MRC) activity in OA chondrocytes shows a significant decrease in complexes II and III compared to normal chondrocytes. On the other hand, mitochondrial mass is increased in OA, as demonstrated by a significant rise in CS activity. Furthermore, OA cells show a reduction in the mitochondrial membrane potential (deltapsim) as demonstrated by using the fluorescent probe JC-1. OA cartilage contains high number of apoptotic chondrocytes, and mitochondria play a key role in apoptosis. Interestingly, OA cartilages show markedly elevated Bcl-2 and caspasa-3 expression. This expression is also correlated with chondrocyte apoptosis and OA lesions. The pathogenesis of OA includes elaboration of increased amounts of NO as a consequence of up-regulation of chondrocyte-inducible NO synthase induced by IL-1, TNF-alpha and other factors. NO reduces chondrocyte survival and induces cell death with morphologic changes characteristic of chondrocyte apoptosis. NO reduces the activity of complex IV and decreases the deltapsim as measured as the ratio of red/green fluorescence. Furthermore, NO induces the mRNA expression of caspase-3 and -7, and it reduces the expression of mRNA bcl-2 and the bcl-2 protein synthesis. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix. Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Direct suppression of mitochondrial respiration promoted MV-mediated mineralization in chondrocytes. Regulation of MRC may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization. After age 40, the incidence of OA in humans increases progressively with increasing age. Studies show a trend to statistic significance between the age and the reduction of complex I activity of human normal chondrocytes. However, the study of relation between age and deltapsim in normal chondrocytes do not demonstrate any significant correlation. It has been reported that as the number of population doublings increased, mitochondrial DNA was degraded and the number of mitochondria per chondrocyte decline. One approach for determining the role of mitochondria in OA is to determine the effects of the MRC inhibition and to compare them with the findings in OA. Inhibition of MRC with antimycin prevents the normal ability of TGFbeta to increase excretion of Pi, thereby worsening deposition of pathologic HA crystals. In chondrocytes, the inhibition of complex IV with NaN3 modified both the deltapsim and the survival of cells inducing apoptosis. Inhibition of complex I with rotenone increases the expression and synthesis of Bcl-2 and Cox-2, both effects are similar effects to produced by IL-1 in human chondrocytes.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPα), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPα gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPα expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPα on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.     

PDK1 promotes apoptosis of chondrocytes via modulating MAPK pathway in osteoarthritis

3-Phosphoinositide dependent protein kinase-1 (PDK1), a serine threonine kinase, belongs to the AGC kinase family and is associated with apoptosis. The aim of this study was to investigate the expression of PDK1 (3-Phosphoinositide dependent protein kinase-1) in articular cartilage with osteoarthritis (OA) and to analyze the relationship between PDK1 and chondrocyte apoptosis. Immunohistochemistry and RT-PCR analysis showed that the expression of PDK1 in articular cartilage of OA patients and healthy controls. IL-1β-stimulated SW1353 cells were used to imitate the OA-like chondrocyte injury in vitro, and IL-1β-induced the expression of PDK1, apoptotic markers(PARP, caspase-3), and phosphorylated p38 were detected by Western blot. The co-localization of PDK1 and Cleaved-caspase3 was confirmed through immunofluorescence. Knocking down PDK1 expression through PDK1 siRNA. Western blot was performed to detect the knockdown efficiency of PDK1 and the impact of PDK1 knockout on IL-1β-induced expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Flow Cytometry-Based Annexin V/PI Staining was used to exam chondrocyte apoptosis. Our experimental results suggested that PDK1 may promote chondrocyte apoptosis in OA via p38 MAPK signaling pathway.     

Prostaglandin PGE2 at very low concentrations suppresses collagen cleavage in cultured human osteoarthritic articular cartilage: this involves a decrease in expression of proinflammatory genes, collagenases and COL10A1, a gene linked to chondrocyte hypertrophy

Suppression of type II collagen (COL2A1) cleavage by transforming growth factor (TGF)-β2 in cultured human osteoarthritic cartilage has been shown to be associated with decreased expression of collagenases, cytokines, genes associated with chondrocyte hypertrophy, and upregulation of prostaglandin (PG)E₂ production. This results in a normalization of chondrocyte phenotypic expression. Here we tested the hypothesis that PGE₂ is associated with the suppressive effects of TGF-β2 in osteoarthritic (OA) cartilage and is itself capable of downregulating collagen cleavage and hypertrophy in human OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with a wide range of concentrations of exogenous PGE₂ (1 pg/ml to 10 ng/ml). COL2A1 cleavage was measured by ELISA. Proteoglycan content was determined by a colorimetric assay. Gene expression studies were performed with real-time PCR. In explants from patients with OA, collagenase-mediated COL2A1 cleavage was frequently downregulated at 10 pg/ml (in the range 1 pg/ml to 10 ng/ml) by PGE₂ as well as by 5 ng/ml TGF-β2. In control OA cultures (no additions) there was an inverse relationship between PGE₂ concentration (range 0 to 70 pg/ml) and collagen cleavage. None of these concentrations of added PGE₂ inhibited the degradation of proteoglycan (aggrecan). Real-time PCR analysis of articular cartilage from five patients with OA revealed that PGE₂ at 10 pg/ml suppressed the expression of matrix metalloproteinase (MMP)-13 and to a smaller extent MMP-1, as well as the proinflammatory cytokines IL-1β and TNF-α and type X collagen (COL10A1), the last of these being a marker of chondrocyte hypertrophy. These studies show that PGE₂ at concentrations much lower than those generated in inflammation is often chondroprotective in that it is frequently capable of selectively suppressing the excessive collagenase-mediated COL2A1 cleavage found in OA cartilage. The results also show that chondrocyte hypertrophy in OA articular cartilage is functionally linked to this increased cleavage and is often suppressed by these low concentrations of added PGE₂. Together these initial observations reveal the importance of very low concentrations of PGE₂ in maintaining a more normal chondrocyte phenotype.     

MFG-E8 regulated by miR-99b-5p protects against osteoarthritis by targeting chondrocyte senescence and macrophage reprogramming via the NF-κB pathway

Milk fat globule-epidermal growth factor (EGF) factor 8 (MFG-E8), as a necessary bridging molecule between apoptotic cells and phagocytic cells, has been widely studied in various organs and diseases, while the effect of MFG-E8 in osteoarthritis (OA) remains unclear. Here, we identified MFG-E8 as a key factor mediating chondrocyte senescence and macrophage polarization and revealed its role in the pathology of OA. We found that MFG-E8 expression was downregulated both locally and systemically as OA advanced in patients with OA and in mice after destabilization of the medial meniscus surgery (DMM) to induce OA. MFG-E8 loss caused striking progressive articular cartilage damage, synovial hyperplasia, and massive osteophyte formation in OA mice, which was relieved by intra-articular administration of recombinant mouse MFG-E8 (rmMFG-E8). Moreover, MFG-E8 restored chondrocyte homeostasis, deferred chondrocyte senescence and reprogrammed macrophages to the M2 subtype to alleviate OA. Further studies showed that MFG-E8 was inhibited by miR-99b-5p, expression of which was significantly upregulated in OA cartilage, leading to exacerbation of experimental OA partially through activation of NF-κB signaling in chondrocytes. Our findings established an essential role of MFG-E8 in chondrocyte senescence and macrophage reprogramming during OA, and identified intra-articular injection of MFG-E8 as a potential therapeutic target for OA prevention and treatment.Subject terms: Predictive markers, Osteoarthritis, Pathogenesis