Role of melatonin combined with exercise as a switch-like regulator for circadian behavior in advanced osteoarthritic knee.
ABSTRACT: Here, we show the role of melatonin combined with or without exercise as a determinant of multicellular behavior in osteoarthritis. We address the relationship between the molecular components governing local circadian clock and changes in the osteoarthritic musculoskeletal axis. Melatonin was injected subcutaneously in animals with advanced knee osteoarthritis (OA) for 4 weeks. Concurrently, moderate treadmill exercise was applied for 30 min/day. Morphometric, histological, and gene/protein-level analyses were performed in the cartilage, synovium, bone, and gastrocnemius muscle. Primary cultured chondrocytes repeatedly exposed to TNF-? were used in an in vitro study. The symptoms of OA include gait disturbance, osteophyte formation, and abnormal metabolism of the extracellular matrix (ECM) of the cartilage. Low-level expression of clock genes was accompanied by aberrant changes in cartilage specimens. Nanomolar doses of melatonin restored the expression of clock-controlled genes and corrected the abnormal chondrocyte phenotype. Melatonin combined with or without exercise prevented periarticular muscle damage as well as cartilage degeneration. But prolonged melatonin administration promoted the proteolytic cleavage of RANKL protein in the synovium, leading to severe subchondral bone erosion. These musculoskeletal changes apparently occurred via the regulation of molecular clock components, suggesting a role of melatonin as a switch-like regulator for the OA phenotype.
Project description:OBJECTIVES: Abnormal chondrocyte gene expression promotes osteoarthritis (OA) pathogenesis. RNA-sequencing revealed that circadian rhythm pathway and expression of core clock protein cryptochrome 2 (Cry2) are dysregulated in human OA cartilage. Here we determined expression patterns and function Cry1 and Cry2. METHODS: Cry mRNA and protein expression was analyzed in normal and OA human and mouse cartilage. Mice with deletion of Cry1 or Cry2 were analyzed for severity of experimental OA and to determine genes and pathways that are regulated by CRY. RESULTS: In human OA cartilage, CRY2 but not CRY1 staining and mRNA expression was significantly decreased. Cry2 was also suppressed in mice with surgical or aging-related OA. Cry2 KO but not Cry1 KO mice with experimental OA showed significantly increased severity of histopathological changes in cartilage, subchondral bone and synovium. In OA chondrocytes, the levels of Cry1 and Cry2 and the amplitude of circadian fluctuation were significantly lower. RNA-seq on knee articular cartilage of wild-type and Cry2 KO mice identified 53 differentially expressed genes, including known CRY2 target circadian genes Nr1d1, Nr1d2, Dbp and Tef. Pathway analysis indicated that circadian rhythm and extracellular matrix remodeling were dysregulated in Cry2 KO mice. CONCLUSIONS: These results show an active role of the circadian clock in general, and of CRY2 in particular, in maintaining ECM homeostasis in cartilage. This cell autonomous network of circadian rhythm genes is disrupted in OA chondrocytes. Targeting CRY2 has potential to correct abnormal gene expression patterns and reduce the severity of OA. Overall design: We report differentially expressed genes between CRY2-KO and WT mouse articular cartilage samples. Mouse articular cartilage was collected from both sides of the femoral condyle and tibial plateau from 3-month-old WT and CRY2-KO mice.
Project description:Arthritis is the leading cause of disability among adults, while osteoarthritis (OA) is the most common form of arthritis that results in cartilage loss. However, accumulating evidence suggests that the protective hyaline cartilage should not be the sole focus of OA treatment. Particularly, synovium also plays essential roles in OA's initiation and progression and warrants serious consideration when battling against OA. Thus, biomarkers with similar OA-responsive expressions in cartilage and synovium should be the potential targets for OA treatment. On the other hand, molecules with a distinguished response during OA in cartilage and synovium should be ruled out as OA therapeutic(s) to avoid controversial effects in different tissues. Here, to pave the path for developing a new generation of OA therapeutics, two published transcriptome datasets of knee articular cartilage and synovium were analyzed in-depth. Genes with statistically significantly different expression in OA and healthy cartilage were compared with those in the synovium. Thirty-five genes with similar OA-responsive expression in both tissues were identified while recognizing three genes with opposite OA-responsive alteration trends in cartilage and synovium. These genes were clustered based on the currently available knowledge, and the potential impacts of these clusters in OA were explored.
Project description:To address the hypothesis that different types of established osteoarthritis (OA) pain behaviours have associations with different aspects of articular pathology, we investigated the relationship between structural knee joint pathology and pain behaviour following injection of a low vs a high dose of monosodium iodoacetate (MIA) in the rat.Rats received a single intra-articular injection of 0.1 mg or 1 mg MIA or saline (control). Pain behaviour (hind limb weight bearing asymmetry (WB) and hindpaw withdrawal threshold (PWT) to punctate stimulation) was assessed. Cartilage and synovium were examined by macroscopic visualisation of articular surfaces and histopathology.Both doses of MIA lowered PWTs, 1 mg MIA also resulted in WB asymmetry. Both doses were associated with cartilage macroscopic appearance, proteoglycan loss, abnormal chondrocyte morphology, increased numbers of vessels crossing the osteochondral junction, synovitis and macrophage infiltration into the synovium. PWTs were more strongly associated with chondrocyte morphology, synovitis and macrophage infiltration than with loss of cartilage surface integrity.Both pain behaviours were associated with OA structural severity and synovitis. Differences in pain phenotype following low vs higher dose of MIA were identified despite similar structural pathology. OA structural pathology as traditionally measured only partially explains the MIA-induced pain phenotype.
Project description:Rheumatoid arthritis (RA) and osteoarthritis (OA) are joint disorders that cause major public health problems. Previous studies of the etiology of RA and OA have implicated Wnt genes, although the exact nature of their involvement remains unclear. To further clarify the relationship between RA, OA, and the Wnt gene family, gene expression analyses were performed on articular cartilage, bone, and synovial tissues in knee joints taken from RA, OA, and nor-mal/control patients. Cytokine assays were also performed in cells transfected with Wnt-7b, a member of the gene family most closely linked to RA and OA. Of the human Wnt genes, real-time PCR analysis revealed significant up-regulation of Wnt-7b in OA cartilage and RA synovium. In situ hybridization and immunohistochemistry also revealed that Wnt-7b was present in articular cartilage, bone, and synovium of RA samples and in osteophytes, articular cartilage, bone marrow, and synovium of OA samples. The levels of the cytokines tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were significantly increased in RA synovium and Wnt-7b-transfected normal synovial cells when compared with normal samples. These results point to the potential involvement of Wnt signaling in the pathobiology of both RA and OA.
Project description:Cartilage destruction in osteoarthritis (OA) is thought to be mediated by two main enzyme families; the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, whereas enzymes from the 'a disintegrin and metalloproteinase domain with thrombospondin motifs' (ADAMTS) family mediate cartilage aggrecan loss. Tissue inhibitors of metalloproteinases (TIMPs) regulate the activity of these enzymes. Although cartilage destruction in OA might be driven by the chondrocyte, low-grade synovitis is reported in patients with all grades of this disease. Our earlier work profiling these gene families in cartilage identified a number of genes that are regulated in OA, which are hence implicated in the disease process. Because the synovium might contribute to cartilage-matrix destruction in OA, we have extended the screening in the current study. We have profiled MMP, ADAMTS and TIMP genes in both cartilage and synovium from patients with either OA of the hip or a fracture to the neck of femur (NOF), giving a more complete picture of proteolysis in this disease. The four most significantly upregulated genes (P < 0.0001) in OA synovium compared to the fractured NOF are MMP28, ADAMTS16, ADAMTS17 and TIMP2. For MMP9, MMP10, MMP12, MMP17, MMP23, MMP28, ADAMTS4, and ADAMTS9, there is a significant correlation between expression levels in the synovium and cartilage, suggesting similar mechanisms of regulation. Additionally, we have shown that in cartilage the median level of steady-state mRNA for MMP13 is approximately 20-fold higher than MMP28 and approximately 1,500-fold higher than ADAMTS16, with expression of this latter gene approximately 150-fold higher in synovium than cartilage. This study is the most comprehensive analysis of the metzincin family of proteinases in the joint to date and has identified several proteinase genes not previously reported to be expressed or regulated in synovium.
Project description:Our study compared the effects of extracorporeal shockwave therapy (ESWT) on the subchondral bone and the articular cartilage in the treatment of early osteoarthritis (OA) of rat knee. The rats were divided into 5 groups which included Sham group, Meniscus group (ESWT applied on medial meniscus), OA group (arthrotomy and medial menisectomy (MMx) and anterior cruciate ligament transection (ACLT), T(M) group (arthrotomy and MMx and ACLT followed by ESWT on medial tibial subchondral bone) and Articular cartilage group (arthrotomy and MMx and ACLT followed by ESWT on medial articular cartilage). Evaluations included the pathological changes of the synovium, articular cartilage and subchondral bone, and compared with ESWT on the meniscus, medial tibial subchondral bone and articular cartilage. The ESWT (0.25 mJ/mm² and 800 impulses) did not cause any damages on the cartilage of the meniscus and the tissue of the joint when compared with Sham group. Among the treatment of osteoarthritic groups (OA, T(M) and Articular cartilage groups), T(M) group showed significant in pathological examination, micro-CT analysis, cartilage grading score and grading of synovium changes by compared with OA and Articular cartilage groups (P < 0.05) in the treatment of early OA knee. In immunohistochemical analysis, T(M) group significantly increased the expression of TGF-?1 but reduced DMP-1, MMP-13 and ADAMTS-5 in the cartilage by compared with OA group and Articular cartilage group (P < 0.05). Our results showed that subchondral bone was an excellent target than articular cartilage for ESWT on early knee osteoarthritis.
Project description:Osteoarthritis (OA) is characterized by the progressive destruction of articular cartilage, which is involved in the imbalance between extracellular matrix (ECM) synthesis and degradation. MicroRNA-140-5p (miR-140) is specifically expressed in cartilage and plays an important role in OA-induced matrix degradation. The aim of this study was to investigate (1) whether intra-articular injection of melatonin could ameliorate surgically induced OA in mice and (2) whether melatonin could regulate matrix-degrading enzymes at the posttranscriptional level by targeting miR-140. In an in vitro OA environment induced by interleukin-1 beta (IL-1?), melatonin treatment improved cell proliferation of human chondrocytes, promoted the expression of cartilage ECM proteins (e.g., type II collagen and aggrecan), and inhibited the levels of IL-1?-induced proteinases, such as matrix metalloproteinase 9 (MMP9), MMP13, ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), and ADAMTS5. Both the microarray and polymerase chain reaction (PCR) experiments revealed that miR-140 was a melatonin-responsive microRNA and melatonin upregulated miR-140 expression, which was suppressed by IL-1? stimulation. In vivo experiments demonstrated that intra-articular injection of melatonin prevented disruptions of cartilage matrix homeostasis and successfully alleviated the progression of surgery-induced OA in mice. Transfection of miR-140 antagomir completely counteracted the antiarthritic effects of melatonin by promoting matrix destruction. Our findings demonstrate that melatonin protects the articular cartilage from OA-induced degradation by targeting miR-140, and intra-articular administration of melatonin may benefit patients suffering from OA.
Project description:Osteoarthritis (OA) is a degenerative disorder characterized by chondrocyte apoptosis and degeneration of articular cartilage resulting in loss of mobility and pain. Inflammation plays a key role in the development and progression of OA both on the side of apoptosis and repair, while its exact role in pathogenesis has yet to be fully elucidated. Few studies have examined the cellular composition (inflammatory cells and/or progenitor cells) in the synovium of patients with pre-OA (asymptomatic with cartilage damage). Therefore, in the current study, mesenchymal progenitor cells (MPCs) and macrophages were enumerated within normal, pre-OA and OA synovium. No differences were observed between MPCs in normal vs. pre-OA, however, fewer macrophages were observed in pre-OA vs. normal synovium. Osteoarthritic synovium contained greater numbers of both MPCs and macrophages. Interestingly, the localization of MPCs and macrophages was affected by disease severity. In normal and pre-OA synovium, MPCs and macrophages co-localized, while in OA synovium, MPCs and macrophage populations were spatially distinct. Examining the cellular interactions between MPCs and macrophages in synovium may be essential for understanding the role of these cells in the onset and/or pathogenesis of the disease. This study has provided a first step by examining these cell types both spatially and temporally (e.g., disease severity). Further cellular and molecular studies will be needed to determine the functions of these cells in the context of disease and in relation to each other and the joint as a whole.
Project description:Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older individuals. Previous studies have shown that over-expression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, it remains unclear which transcription factors regulate the expression of these cartilage-degrading molecules in articular chondrocytes. This study demonstrated that mice lacking Nfat1, a member of the nuclear factor of activated T cells (NFAT) transcription factors, exhibited normal skeletal development but displayed loss of type II collagen (collagen-2) and aggrecan with over-expression of specific matrix-degrading proteinases and proinflammatory cytokines in young adult articular cartilage of load-bearing joints. These initial changes are followed by articular chondrocyte proliferation/clustering, progressive articular surface destruction, periarticular chondro-osteophyte formation and exposure of thickened subchondral bone, all of which resemble human OA. Forced expression of Nfat1 delivered with lentiviral vectors in cultured 3 month-old primary Nfat1 knockout (Nfat1(-/-)) articular chondrocytes partially or completely rescued the abnormal catabolic and anabolic activities of Nfat1(-/-) articular chondrocytes. These new findings revealed a previously unrecognized critical role of Nfat1 in maintaining the physiological function of differentiated adult articular chondrocytes through regulating the expression of specific matrix-degrading proteinases and proinflammatory cytokines. Nfat1 deficiency causes OA due to an imbalance between the catabolic and anabolic activities of adult articular chondrocytes, leading to articular cartilage degradation and failed repair activities in and around articular cartilage. These results may provide new insights into the aetiology, pathogenesis and potential therapeutic strategies for osteoarthritis.
Project description:Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA.