Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions.

We investigated the effects of hypoxia-inducible factor (HIF)-1α on articular cartilage under mechanical stimulation and the associated mechanisms. Chondrocytes, isolated from articular cartilage from the knee, hip, and shoulder joints of Wistar rats, were subjected to 20 % tensile stress under hypoxic (5% O2) conditions for 24 h. HIF-1α and aggrecan expression was significantly enhanced with mechanical stimulation under hypoxia but not significantly altered with mechanical stimulation under normoxia. The nuclear translocation of HIF-1α was enhanced by mechanical stress under hypoxia. Under both normoxia and hypoxia, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) 5 expression was significantly reduced with mechanical stimulation compared to that in the group without mechanical stimulation. However, HIF-1α knockdown mitigated changes in aggrecan and ADAM-TS5 expression mediated by mechanical stimulation under hypoxia. The effects of treadmill running on HIF-1α production in the articular cartilage of rat knee joints were also analyzed. HIF-1α production increased in the moderate running group and decreased to the same levels as those in the control group in the excessive running group. This suggests that HIF-1α regulates aggrecan and ADAM-TS5 expression in response to mechanical stimulation under hypoxia and general mechanical stimulation in articular cartilage under hypoxia, while controlling cartilage homeostasis.

NF-Ä¸ß upregulates ADAMTS5 expression by direct binding after TNF-α treatment in OUMS-27 chondrosarcoma cell line.

Inflammation caused-aggrecan degradation is a critical event in the pathogenesis of osteoarthritis (OA). The aggrecanases like a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) are assumed to be key players in the aggrecan destruction. To develop the comprehensive therapy method for OA, it is essential to elucidate the activation mechanism of ADAMTS5 gene after stimulation of inflammatory cytokines like tumor necrosis factor-α (TNF-α). The cell lines of human chondrosarcoma (OUMS-27) and embryonic kidney (HEK293T) were incubated with tumor necrosis factor-α (TNF-α) for certain time periods, and the expression level of ADAMTS5 was measured in both mRNA and protein levels. Tissue-specific ADAMTS5 activation was founded to be induced after TNF-α treatment. Then, the constructs for the promoter region of ADAMTS5 were prepared and luciferase assay was conducted to understand the involvement mechanism of nuclear factor-kappa beta (NF-ĸß) in ADAMTS5 activation. It was demonstrated that NF-Ä¸ß induces the ADAMTS5 expression level by directly binding the promoter region of ADAMTS5. Although the TNF-α blocker is used for OA treatment, the development of a more comprehensive treatment strategy is an urgent need. Our experimental data contributes in terms of selecting NF-Ä¸ß as a target molecule. Up to date, NF-Ä¸ß has been proven to involve in the ADAMTS5 up-regulation after several pro-inflammatory cytokines stimulation. In conclusion, our findings make important contributions to the knowledge about the roles of NF-Ä¸ß in ADAMTS5 activation under inflammatory conditions. So, NF-Ä¸ß could be considered to be a potential target for OA treatment.

Periostin Mediates Condylar Resorption via the NF-κB-ADAMTS5 Pathway.

Although the up-regulation of periostin in osteoarthritic (OA) is found, its function on OA condyle caused by disc displacement is not clear. Our objective was to explore whether periostin has any effect on condylar resorption. We initially identified periostin-positive cells in temporomandibular joint osteoarthritic (TMJ-OA) cartilage. Furthermore, the vitro analysis confirmed that the expression of periostin in chondrocytes treated with a static pressure of 150 kpa and 200 kpa for 3 h by an in-house-designed pressure chamber. To explore the underlying mechanism, we found that periostin can induce IκBα phosphorylation and its subsequent degradation, leading to consequent p65 nuclear translocation and subsequent induction of ADAMTS5 expression, which is known to be detrimental to cartilage extracellular matrix production. Importantly, inhibiting NF-κB signaling, by BAY 11-7082 treatment, rescued periostin-induced ADAMTS5 up-regulation. This study elucidated the direct role of periostin in condylar resorption, which was found to occur via NF-κB-ADAMTS5 signaling. Inhibition of this pathway might provide a new strategy for TMJ-OA treatment.

Leonurine Hydrochloride Suppresses Inflammatory Responses and Ameliorates Cartilage Degradation in Osteoarthritis via NF-κB Signaling Pathway.

Leonurine hydrochloride (LH) has been reported to exhibit a number of biological properties such as suppression of inflammation. This study aimed to examine whether the progression of osteoarthritis (OA) could be delayed by the administration of LH in an OA model. Rat chondrocytes were treated with LH under the condition of TNF-α-induced inflammation. After that, real-time PCR and Western blotting were conducted to evaluate relative gene/protein expression levels. For the in vivo study, rats were randomly allocated to a control group (anterior cruciate ligament transection (ACLT) surgery, treatment with saline) and LH group (ACLT surgery, treatment with LH). Articular cartilage degeneration was assessed by histological evaluation. It was found that LH significantly suppressed the expression of MMP-1, MMP-3, MMP-13, IL-6, and ADAMTS-5 in cells via the NF-κB signaling pathway. In addition, it is revealed that intra-articular injection of LH significantly ameliorated cartilage degeneration in a rat OA model. Taken together, these results indicate that LH attenuates progression of OA by inhibition of inflammation via the NF-κB signaling pathway and represents a potential preventive therapy for OA.

Evaluating the Effects of Platelet-Rich Plasma and Amniotic Viscous Fluid on Inflammatory Markers in a Human Coculture Model for Osteoarthritis.

PURPOSE: To assess the anti-inflammatory effects of platelet-rich plasma (PRP) and amniotic viscous fluid using a human coculture system of cartilage and synovial tissue from osteoarthritic patients. METHODS: A coculture system was created using cartilage and synovium from 3 patients undergoing total knee arthroplasty. To induce inflammation, interleukin-1ß was added to each coculture. Biologic agents tested included 2 PRP concentrations (PRPL and PRPH) and 2 different samples of amniotic viscous fluid (Amnion and Flograft). Amnion was also tested with PRP to check for any additive effects. Quantitative polymerase chain reaction was used to measure gene expression of factors involved in osteoarthritis, including disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), tissue inhibitor of metalloproteinases 1 (TIMP-1), vascular endothelial growth factor (VEGF), aggrecan, type 1 collagen, and nitric oxide, at 0, 24, 48, and 72 hours. A synthetic nonsteroidal medication, Ketorolac, was used for baseline comparison to the biologic agents. RESULTS: When comparing from time 0, both Amnion and Flograft resulted in significant decreases of ADAMTS-5 and TIMP-1 gene expression in cartilage and synovium for up to 72 hours. Both amniotic preparations increased collagen-1 gene expression in cartilage and decreased VEGF expression in synovium. Amnion was not found to have any effect on nitric oxide concentration at any time point (P > .05), as opposed to both PRP concentrations (P < .05). All biologic agents showed differences in gene expression similar to Ketorolac in ADAMTS-5, TIMP-1, and VEGF expression. CONCLUSION: This study found that amniotic fluid had anti-inflammatory effects mostly similar to those of both PRPH and PRPL; however, no significant additive effects in reducing inflammatory gene expression were found when combining biologic agents. CLINICAL RELEVANCE: PRP and amniotic fluid may provide alternative treatment options to delay the progression of the disease without the systemic and intra-articular side effects of corticosteroids.

ADAMTS5 acts as a tumor suppressor by inhibiting migration, invasion and angiogenesis in human gastric cancer.

BACKGROUND: ADAMTS5 has been reported to be involved in the progression of several human tumors. Nevertheless, the role of ADAMTS5 in gastric cancer (GC) remains poorly defined. METHODS: ADAMTS5 expression levels were analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) in GC cell lines and tissues, and the correlations between ADAMTS5 expression and clinicopathological features and survival were also examined. In vitro assays, including transwell assays, wound healing assays and cell adhesion assays, were employed to further explore the biological functions of ADAMTS5. A MAP kinase pathway microarray was used to identify the underlying mechanisms. The expression of ADAMTS5 and ETS1 and the microvessel density (MVD) were also analyzed using IHC to determine correlations with angiogenesis in GC. RESULTS: ADAMTS5 expression was downregulated in gastric cancer tissues. Low expression of ADAMTS5 was associated with gender, histological type, degree of differentiation, M stage, TNM stage and vascular invasion, and was also an independent indicator of a poor prognosis for patients with GC. ADAMTS5 overexpression markedly inhibited GC cell migration and invasion and enhanced cell adhesion to the extracellular matrix (ECM), whereas knockdown of ADAMTS5 exerted the opposite effects. Furthermore, the ADAMTS5 expression status was negatively correlated with ETS1 expression and MVD. CONCLUSION: ADAMTS5 is downregulated in GC and suppresses tumor metastasis and angiogenesis by inhibiting ETS1-mediated changes in MVD and potentially acts as a novel prognostic marker and a potential therapeutic target in human GC.

Melatonin Prevents Osteoarthritis-Induced Cartilage Degradation via Targeting MicroRNA-140.

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-1ß), 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-1ß-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-1ß 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.

MiR-132-3p regulates ADAMTS-5 expression and promotes chondrogenic differentiation of rat mesenchymal stem cells.

Previous study showed that miRNA aberrant expression is involved in chondrogenic differentiation. In this study, we aimed to investigate the effects of miR-132-3p on chondrogenic differentiation and the underlying mechanisms. First, quantitative PCR were performed to determine the level of MiR-132-3p. Then, we used luciferase assay to examine the target of miR-132-3p. Proteoglycan was tested by Alcian blue staining assay. Moreover, the sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) levels were analyzed by quantitative PCR, immunofluorescence and Western blotting. Our results showed that MiR-132-3p level was reduced in rat MSCs (rMSCs) during chondrogenic differentiation. Ectopic expression of miR-132-3p induced proteoglycan accumulation and the increase of ACAN, SOX9 and COL2A1 expression, which were involved in inducing chondrogenic differentiation of rMSCs. More importantly, ADAMTS-5 was identified as the target of MiR-132-3p. Knockdown of ADAMTS-5 increased proteoglycan level, but reduced the SOX9, ACAN, and COL2A1 levels during chondrogenic differentiation of rMSCs. Taken together, our results revels that MiR-132-3p promotes rMSCs chondrogenic differentiation, possibly mediated by targeting ADAMTS-5, which provided new perspective on the chondrogenic differentiation and pathology of osteoarthritis.

Epigenetic silencing of ADAMTS5 is associated with increased invasiveness and poor survival in patients with colorectal cancer.

PURPOSE: A disintegrin and metalloprotease with motif 5(ADAMTS5) has been involved in colorectal cancer (CRC) with hypermethylation in the promoter. However, its role in CRC remains unclear. The aim of this study was to explore the clinical significance and biological effect of ADAMTS5 on colorectal carcinogenesis. Through MSP, qRT-PCR, WB and IHC analysis, followed by a variety of in vitro assays, we report the function of ADAMTS5 in CRC. ADAMTS5 was markedly hypermethylaed and downregulated in tumor tissues compared with non-tumor tissues (p < 0.001). Negative expression of ADAMTS5 was much more common in tumor tissues than that in normal tissues (p < 0.001) and correlated with histologic types (p = 0.002), poor OS (p = 0.029) and DFS (p = 0.018). In vitro assay revealed that overexpression of ADAMTS5 inhibited the capabilities of migration and invasion of CRC cells, and no effect on cell growth, cell cycle and apoptosis. ADAMTS5 is hypermethylated and inhibits cancer cells invasion and migration in colorectal cancer, and correlates with OS and DFS, indicating that ADAMTS5 might be a useful biomarker in colorectal cancer therapy.

Adamts 1, 4, 5, 8, and 9 in Early Pregnancies.

INTRODUCTION: The aim of this study was to immunohistochemically investigate the presence and localization of ADAMTS 1, 4, 5, 8 and 9 in decidual and chorionic tissues in first trimester pregnancy losses. MATERIALS AND METHODS: This study was conducted with early pregnancy failure decidual and chorionic tissue samples from 36 pregnant women in the first trimester of pregnancy (ongoing pregnancies, missed miscarriages, anembryonic pregnancies) Results: It was observed that the decidual and chorionic tissue levels of ADAMTS 1, 4, 5, and 8 in ongoing pregnancies were more intensely expressed when compared with miscarriages. ADAMTS 1 expression was not observed in the anembryonic pregnancies, ADAMTS 4, 5, and 8 were less intensely expressed. ADAMTS 9 showed no staining in any group. CONCLUSION: ADAMTS 1 may be necessary during the decidualization and implantation stages of early normal pregnancy.

Intra-articular injection of microRNA-140 (miRNA-140) alleviates osteoarthritis (OA) progression by modulating extracellular matrix (ECM) homeostasis in rats.

OBJECTIVE: Disruptions of extracellular matrix (ECM) homeostasis are key events in the pathogenesis of osteoarthritis (OA). MicroRNA-140 (miRNA-140) is expressed specifically in cartilage and regulates ECM-degrading enzymes. Our objective in this study was to determine if intra-articular injection of miRNA-140 can attenuate OA progression in rats. DESIGN: miRNA-140 levels in human normal and OA cartilage derived chondrocytes and synovial fluid were assessed by polymerase chain reaction (PCR). After primary human chondrocytes were transfected with miRNA-140 mimic or inhibitor, PCR and western blotting were performed to quantify Collagen II, MMP-13, and ADAMTS-5 expression. An OA model was induced surgically in rats, and subsequently treated with one single intra-articular injection of miRNA-140 agomir. At 4, 8, and 12 weeks after surgery, OA progression were evaluated macroscopically, histologically, and immunohistochemically in these rats. RESULTS: miRNA-140 levels were significantly reduced in human OA cartilage derived chondrocytes and synovial fluid compared with normal chondrocytes and synovial fluid. Overexpressing miRNA-140 in primary human chondrocytes promoted Collagen II expression and inhibited MMP-13 and ADAMTS-5 expression. miRNA-140 levels in rat cartilage were significantly higher in the miRNA-140 agomir group than in the control group. Moreover, behavioural scores, chondrocyte numbers, cartilage thickness and Collagen II expression levels in cartilage were significantly higher, while pathological scores and MMP-13 and ADAMTS-5 expression levels were significantly lower in the miRNA-140 agomir group than in the control group. CONCLUSION: Intra-articular injection of miRNA-140 can alleviate OA progression by modulating ECM homeostasis in rats, and may have potential as a new therapy for OA.

ADAMTS5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration.

STUDY DESIGN: ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. SUMMARY OF BACKGROUND DATA: We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. METHODS: Three-month-old WT (C57BL/6) and ADAMTS5 mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. RESULTS: Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. CONCLUSION: The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. LEVEL OF EVIDENCE: N/A.

In vitro effects of triamcinolone acetonide and in combination with hyaluronan on canine normal and spontaneous osteoarthritis articular cartilage.

The purposes of this study were to examine the cartilage degradation effects of triamcinolone acetonide (TA) on normal and osteoarthritic (OA) primary canine chondrocytes and cartilage explants and to examine the cartilage degradation effects of TA in combination with low-molecular-weight hyaluronan (LMWHA). To assess the effects of these drugs on cell culture, 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and real-time PCR were used to measure chondrotoxicity and determine gene expression, respectively. Uronic acid and hydroxyproline remaining in cartilage and histopathology were used to estimate the effects of these drugs on cartilage explants. In chondrocyte cultures, TA reduced chondrocyte viability in a concentration-dependent manner. LMWHA 2.5 mg/ml combined with TA at IC20 (0.09 mg/ml) could increase the viability of normal chondrocytes when compared with TA-treated alone. TA at IC20 induced down-regulation of ACAN and induced up-regulation of ADAMTS5 in canine normal chondrocytes. TA at IC20 (0.11 mg/ml) up-regulated ADAMTS5, MMP2, MMP3, MMP13, and ACAN expression in canine OA chondrocytes. In explant culture, TA at 1.25, 2.5, and 5 mg/ml increased the severity of structural damage, chondrocyte loss and cluster formation, and proteoglycan loss in OA cartilage. LMWHA could decrease the chondrotoxicity of TA at IC20 only in normal chondrocytes, as observed by chondrocyte viability. The combination of LMWHA and TA did not show clearly beneficial effects in all other normal and OA samples. Consequently, using TA alone or in combination with LMWHA in OA cartilage should be of concern because it may lead to cartilage destruction.

Role of ADAMTS5 in Unexplained Fetal Growth Restriction (FGR).

AIM: We aim to determine the role of serum and placental A disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5) in fetal growth restriction (FGR). MATERIAL AND METHODS: 43 pregnancies suffering FGR and 45 healthy ones were homogenized for their body mass indices, ages, and gestational weeks. Expression of ADAMTS5 in placental samples was determined by immunohistochemical methods and concurrent maternal serum ADAMTS5 levels were determined with enzyme-linked immunosorbent assay. RESULTS: Expression of ADAMTS5 was higher in FGR group than the healthy control in placenta. Both the cytoplasmic staining pattern of the syncytiotrophoblasts and staining of the decidual plate were shown in the FGR group; but not in the control group. A negative correlation between serum ADAMTS5 levels and birth weight in FGR group was observed. CONCLUSION: Increased ADAMTS5 levels were observed in placental insufficiency cases. This study demonstrates that ADAMTS5 may be a sensitive indicator of placental insufficiency which has variable factors in etiology. Additional work is needed to delineate the mechanism of its involvement.