DNA demethylation of promoter region orchestrates SPI-1-induced ADAMTS-5 expression in articular cartilage of osteoarthritis mice.

Osteoarthritis (OA) is one of the most prevalent joint diseases in aged people and characterized by articular cartilage degeneration, synovial inflammation, and abnormal bone remodeling. Recent advances in OA research have clearly shown that OA development is associated with aberrant DNA methylation status of many OA-related genes. As one of most important cartilage degrading proteases in OA, a disintegrin and metalloproteinase with thrombospondin motifs subtype 5 (ADAMTS-5) is activated to mediate cartilage degradation in human OA and experimental murine OA models. The pathological factors and signaling pathways mediating ADAMTS-5 activation during OA development are not well defined and have been a focus of intense research. ADAMTS-5 promoter is featured by CpG islands. So far there have been no reports concerning the DNA methylation status in ADAMTS-5 promoter during OA development. In this study, we sought to investigate DNA methylation status in ADAMTS-5 promoter, the role of DNA methylation in ADAMTS-5 activation in OA, and the underlying mechanisms. The potential for anti-OA intervention therapy which is based on modulating DNA methylation is also explored. Our results showed that DNA methyltransferases 1 (Dnmt1) downregulation-associated ADAMTS-5 promoter demethylation played an important role in ADAMTS-5 activation in OA, which facilitated SPI-1 binding on ADAMTS-5 promoter to activate ADAMTS-5 expression. More importantly, OA pathological phenotype of mice was alleviated in response to Dnmt1-induced DNA methylation of ADAMTS-5 promoter. Our study will benefit not only for deeper insights into the functional role and regulation mechanisms of ADAMTS-5 in OA, but also for the discovery of disease-modifying OA drugs on the basis of ADAMTS-5 via modulating DNA methylation status.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Inhibitory Effect of a Tankyrase Inhibitor on Mechanical Stress-Induced Protease Expression in Human Articular Chondrocytes.

We investigated the effects of a Tankyrase (TNKS-1/2) inhibitor on mechanical stress-induced gene expression in human chondrocytes and examined TNKS-1/2 expression in human osteoarthritis (OA) cartilage. Cells were seeded onto stretch chambers and incubated with or without a TNKS-1/2 inhibitor (XAV939) for 12 h. Uni-axial cyclic tensile strain (CTS) (0.5 Hz, 8% elongation, 30 min) was applied and the gene expression of type II collagen a1 chain (COL2A1), aggrecan (ACAN), SRY-box9 (SOX9), TNKS-1/2, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), and matrix metalloproteinase-13 (MMP-13) were examined by real-time PCR. The expression of ADAMTS-5, MMP-13, nuclear translocation of nuclear factor-κB (NF-κB), and ß-catenin were examined by immunocytochemistry and Western blotting. The concentration of IL-1ß in the supernatant was examined by enzyme-linked immunosorbent assay (ELISA). TNKS-1/2 expression was assessed by immunohistochemistry in human OA cartilage obtained at the total knee arthroplasty. TNKS-1/2 expression was increased after CTS. The expression of anabolic factors were decreased by CTS, however, these declines were abrogated by XAV939. XAV939 suppressed the CTS-induced expression of catabolic factors, the release of IL-1ß, as well as the nuclear translocation of NF-κB and ß-catenin. TNKS-1/2 expression increased in mild and moderate OA cartilage. Our results demonstrated that XAV939 suppressed mechanical stress-induced expression of catabolic proteases by the inhibition of NF-κB and activation of ß-catenin, indicating that TNKS-1/2 expression might be associated with OA pathogenesis.

Investigating the association between serum ADAM/ADAMTS levels and bone mineral density by mendelian randomization study.

PURPOSE: A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) have been reported potentially involved in bone metabolism and related to bone mineral density. This Mendelian Randomization (MR) analysis was performed to determine whether there are causal associations of serum ADAM/ADAMTS with BMD in rid of confounders. METHODS: The genome-wide summary statistics of four site-specific BMD measurements were obtained from studies in individuals of European ancestry, including forearm (n = 8,143), femoral neck (n = 32,735), lumbar spine (n = 28,498) and heel (n = 426,824). The genetic instrumental variables for circulating levels of ADAM12, ADAM19, ADAM23, ADAMTS5 and ADAMTS6 were retrieved from the latest genome-wide association study of European ancestry (n = 5336 ~ 5367). The estimated causal effect was given by the Wald ratio for each variant, the inverse-variance weighted model was used as the primary approach to combine estimates from multiple instruments, and sensitivity analyses were conducted to assess the robustness of MR results. The Bonferroni-corrected significance was set at P < 0.0025 to account for multiple testing, and a lenient threshold P < 0.05 was considered to suggest a causal relationship. RESULTS: The causal effects of genetically predicted serum ADAM/ADAMTS levels on BMD measurements at forearm, femoral neck and lumbar spine were not statistically supported by MR analyses. Although causal effect of ADAMTS5 on heel BMD given by the primary MR analysis (ß = -0.006, -0.010 to 0.002, P = 0.004) failed to reach Bonferroni-corrected significance, additional MR approaches and sensitivity analyses indicated a robust causal relationship. CONCLUSION: Our study provided suggestive evidence for the causal effect of higher serum levels of ADAMTS5 on decreased heel BMD, while there was no supportive evidence for the associations of ADAM12, ADAM19, ADAM23, and ADAMTS6 with BMD at forearm, femoral neck and lumbar spine in Europeans.

ADAMTS5 promotes neovascularization via autophagic degradation of PEDF in proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) adversely affects visual function. Extracellular matrix proteins (ECM) contribute significantly to the development of PDR. A Disintegrin and Metalloproteinase with Thrombospondin motifs 5 (ADAMTS5) is a member of ECM proteins. ADAMTS5 participates in angiogenesis and inflammation in diverse diseases. However, the role of ADAMTS5 in PDR remains elusive. Multiplex beam array technology was used to analyze vitreous humor of PDR patients and normal people. ELISA and Western blot were used to detect the expression of ADAMTS5, PEDF and autophagy related factors. Immunofluorescence assay was used to mark the expression and localization of ADAMTS5 and PEDF. The neovascularization was detected by tube formation test. Our results revealed that ADAMTS5 expression was increased in the vitreous humor of PDR patients and oxygen-induced retinopathy (OIR) mice retinas. Inhibiting ADAMTS5 alleviated pathological angiogenesis and upregulated PEDF expression in the OIR mice. In addition, ADAMTS5 inhibited PEDF secretion in ARPE-19 cells in vitro studies, thereby inhibiting the migration of HMEC-1. Mechanically, ADAMTS5 promoted the autophagic degradation of PEDF. Collectively, inhibition of ADAMTS5 during OIR suppresses pathological angiogenesis. Our study provides a new approach for resolving pathological angiogenesis in PDR.

LncRNA XIST promotes mitochondrial dysfunction of hepatocytes to aggravate hepatic fibrogenesis via miR-539-3p/ADAMTS5 axis.

A previous study indicated that long non-coding RNA X-inactive-specific transcript (XIST) promoted ethanol-induced HSCs autophagy and activation. Considering the critical role of HSC activation in hepatic fibrosis, the aim of the present study was to reveal the exact role of XIST in liver fibrosis and its underlying mechanism. The expression of XIST in the liver from CCL4-induced mice and control mice as well as human fibrotic liver tissue and healthy liver tissue was examined. The mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potential (MMP), and mitochondrial morphology were measured to assess the mitochondrial damage. The relationship between XIST and miR-539-3p as well as between miR-539-3p and ADAMTS5 was verified by a dual-luciferase reporter assay. The expression levels of HSCs activation markers were examined by Western blot. The results showed that the XIST was upregulated in fibrotic liver tissue, and overexpression of XIST induced mitochondrial dysfunction in hepatocytes. miR-539-3p directly targeted XIST, and ADAMTS5 mRNA was a downstream target of miR-539-3p. Knockdown of miR-539-3p led to an increased mitochondrial damage in hepatocytes in terms of reduced mitochondrial length, decreased MMP, and increased ROS production. However, the depletion of ADAMTS5 reversed the regulatory effect of XIST on mitochondrial damage in hepatocytes and the activation of HSCs. Our study revealed the critical role of the XIST/miR-539-3p/ADAMTS5 axis in regulating mitochondrial damage in hepatocytes and the activation of HSCs. This study may provide a potential therapeutic strategy for the treatment of liver fibrosis.

Concentration-dependent effects of leptin on osteoarthritis-associated changes in phenotype of human chondrocytes.

Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.

The adaptive response of the mandibular condyle to increased load is disrupted by ADAMTS5 deficiency.

OBJECTIVE: To determine the impact of increased load on the temporomandibular joint (TMJ) from mice deficient in the extracellular matrix protease ADAMTS5. MATERIALS AND METHODS: Wire springs exerting 0.5 N for 1 h/day for 5 days (Adamts5+/+ -n = 18; Adamts5-/- n = 19) or 0.8 N for 1 h/day for 10 days (Adamts5+/+-n = 18; Adamts5-/- n = 17) were used to increase murine TMJ load. Safranin O-staining was used to determine mandibular condylar cartilage (MCC) morphology. Chondrogenic factors Sox9 and aggrecan were immunolocalized. Microcomputed topography was employed to evaluate mineralized tissues, and Tartrate-Resistant Acid Phosphatase staining was used to quantify osteoclasts. RESULTS: Increased load on the mandibular condyle of Adamts5-/- mice resulted in an increase in the hypertrophic zone of mandibular condylar cartilage (MCC) compared to normal load (NL) (P < 0.01). In the trabecular bone of the mandibular condyle, the total volume (TV), bone volume (BV), trabecular thickness (TbTh), and trabecular separation (TbSp) of the mandibular condyles in Adamts5-/- mice (n = 27) did not change significantly with increased load, compared to Adamts5+/+ (n = 38) that exhibited significant responses (TV-P < 0.05; BV-P < 0.001; TbTh-P < 0.01; TbSp-P < 0.01). The bone volume fraction (BV/TV) was significantly reduced in response to increased load in both Adamts5-/- (P < 0.05) and Adamts5+/+ mandibular condyles (P < 0.001) compared to NL. Increased load in Adamts5-/- mandibular condyles also resulted in a dramatic increase in osteoclasts compared to Adamts5-/- NL (P < 0.001) and to Adamts5+/+ with increased load (P < 01). CONCLUSION: The trabeculated bone of the Adamts5-/- mandibular condyle was significantly less responsive to the increased load compared to Adamts5+/+. ADAMTS5 may be required for mechanotransduction in the trabeculated bone of the mandibular condyle.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis.

OBJECTIVE: As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-ß signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown. METHODS: Sprague-Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3. RESULTS: In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete. CONCLUSION: The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis.

Skeletal muscle, as a regenerative organization, plays a vital role in physiological characteristics and homeostasis. However, the regulation mechanism of skeletal muscle regeneration is not entirely clear. miRNAs, as one of the regulatory factors, exert profound effects on regulating skeletal muscle regeneration and myogenesis. This study aimed to discover the regulatory function of important miRNA miR-200c-5p in skeletal muscle regeneration. In our study, miR-200c-5p increased at the early stage and peaked at first day during mouse skeletal muscle regeneration, which was also highly expressed in skeletal muscle of mouse tissue profile. Further, overexpression of miR-200c-5p promoted migration and inhibited differentiation of C2C12 myoblast, whereas inhibition of miR-200c-5p had the opposite effect. Bioinformatic analysis predicted that Adamts5 has potential binding sites for miR-200c-5p at 3'UTR region. Dual-luciferase and RIP assays further proved that Adamts5 is a target gene of miR-200c-5p. The expression patterns of miR-200c-5p and Adamts5 were opposite during the skeletal muscle regeneration. Moreover, miR-200c-5p can rescue the effects of Adamts5 in the C2C12 myoblast. In conclusion, miR-200c-5p might play a considerable function during skeletal muscle regeneration and myogenesis. These findings will provide a promising gene for promoting muscle health and candidate therapeutic target for skeletal muscle repair.

Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. METHODS: Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1ß-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. RESULTS: GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and <23 ± 1 nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 µM and 10 µM, respectively). In DMM mice, GLPG1972/S201086 (30-120 mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50 mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). CONCLUSIONS: GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

Excessive downhill training leads to early onset of knee osteoarthritis.

OBJECTIVE: Increased levels of pro-inflammatory cytokines are associated with the release of degradative enzymes leading to osteoarthritis (OA) development. Although physical exercise (PE) is generally recognized as beneficial for OA symptoms, excessive training workload and eccentric muscular exercise have increased OA risk. Here, we investigated the effects of excessive exercise workload and exercise type on systemic inflammation and knee joint OA. METHODS: Mice were divided into five groups: sedentary (SED), uphill training (TRU), downhill training (TRD), excessive uphill training (ETU), and excessive downhill training (ETD) for an 8-week training intervention protocol. RESULTS: ETD group had increased pro-inflammatory cytokines in serum, vastus lateralis (VL), and vastus medialis (VM) muscles, while ETU group mice had increased cytokine levels in the VL and VM. Total knee joint OARSI score were more significant in ETD group compared to SED and TRU groups. They were also more meaningful for the medial tibial plateau of ETD group compared to SED group. MMP-3 and cleaved Caspase-3 were higher in the ETD group than the SED and TRU group, while Adamts-5 was higher in the ETD group than the SED group. TRU group had increased PRG-4 levels compared to ETU and ETD group. ETD group had decreased total bone volume, trabecular bone volume, and cortical thickness compared to SED group. CONCLUSION: Excessive downhill training induced a chronic pro-inflammatory state in mice and was associated with early signs of cartilage and bone degeneration that are clinical indicators of knee OA.

Long non-coding RNA XIST regulates chondrogenic differentiation of synovium-derived mesenchymal stem cells from temporomandibular joint via miR-27b-3p/ADAMTS-5 axis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative disease in jaw joint, accompanied by articular cartilage destruction. Differentiation of stem cells to cartilage has important therapeutic implications in TMJ cartilage repair. Previous studies revealed that lncRNA XIST participated in various biological processes. However, the effect of XIST on chondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs) remains unclear. Our study aimed to investigate the function of XIST in chondrogenic differentiation of human SMSCs from TMJ. METHODS: Alcian blue staining was performed to determine proteoglycan in SMSCs. qPCR, western blotting and immunofluorescence assays were allowed to assess sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) expression. The direct interaction between miR-27b-3p and XIST or ADAMTS-5 was confirmed by dual luciferase reporter assay or RNA immunoprecipitation (RIP) assay. RESULTS: XIST was remarkably down-regulated in chondrogenic differentiation of SMSCs. Functional analysis demonstrated that XIST silencing promoted chondrogenic differentiation of SMSCs. Dual luciferase reporter and RIP assays identified that XIST acted as a sponge for miR-27b-3p. Moreover, XIST regulated ADAMTS-5 expression by directly binding miR-27b-3p. More importantly, miR-27b-3p/ADAMTS-5 rescued the effects of XIST on chondrogenic differentiation of SMSCs. CONCLUSION: The results suggest that XIST modulates SMSCs chondrogenic differentiation via the miR-27b-3p/ADAMTS-5 axis, which provides new targets for TMJOA treatment.

Efficacy of Chondroprotective Food Supplements Based on Collagen Hydrolysate and Compounds Isolated from Marine Organisms.

Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.

IGF-1 Upregulates Biglycan and Decorin by Increasing Translation and Reducing ADAMTS5 Expression.

Proteoglycan (PG) is a glycosaminoglycan (GAG)-conjugated protein essential for maintaining tissue strength and elasticity. The most abundant skin PGs, biglycan and decorin, have been reported to decrease as skin ages. Insulin-like growth factor-1 (IGF-1) is important in various physiological functions such as cell survival, growth, and apoptosis. It is well known that the serum level of IGF-1 decreases with age. Therefore, we investigated whether and how IGF-1 affects biglycan and decorin. When primary cultured normal human dermal fibroblasts (NHDFs) were treated with IGF-1, protein levels of biglycan and decorin increased, despite no difference in mRNA expression. This increase was not inhibited by transcription blockade using actinomycin D, suggesting that it is mediated by IGF-1-induced enhanced translation. Additionally, both mRNA and protein expression of ADAMTS5, a PG-degrading enzyme, were decreased in IGF-1-treated NHDFs. Knockdown of ADAMTS5 via RNA interference increased protein expression of biglycan and decorin. Moreover, mRNA and protein expression of ADAMTS5 increased in aged human skin tissues compared to young tissue. Overall, IGF-1 increases biglycan and decorin, which is achieved by improving protein translation to increase synthesis and preventing ADAMTS5-mediated degradation. This suggests a new role of IGF-1 as a regulator for biglycan and decorin in skin aging process.

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.

The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo.

Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody® (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy-cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1ß (10 ng/mL)) with or without M6495 (1000-0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.

The investigation of serum levels of ADAMTS 5 and 8 (the A disintegrin and metalloproteinase with thrombospondin motifs) in the etiology of endometrial cancer.

The aim of this study was to investigate the serum levels of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 and 8 in patients diagnosed with endometrial cancer. Our study included 41 patients diagnosed with endometrial cancer. The control group consisted of 41 patients diagnosed with benign endometrial pathology. The serum samples were centrifuged and stored at -80 °C. The serum levels of ADAMTS were significantly higher (p<.001), whereas the levels of ADAMTS 8 were significantly lower in patients diagnosed with cancer (p<.001). In addition to the presence of known factors in the aetiology of endometrial cancer, the effect of inflammatory factors and some new proteins has centred on the causes of tumourigenesis in recent years. In this sense, these proteins, called the ADAMTS, are the source of new studies.Impact StatementWhat is already known on this subject? When the recent studies about endometrial cancer are evaluated, it is seen that the effects of chronic inflammation and cytokines have gained importance in its aetiology. The A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) gene family consist of 19 proteases that play essential role in the formation of the extracellular matrix (ECM) and interact with inflammatory cytokines. These proteases and their substrates provide a wide range of functions in different tissues, including ECM remodelling, angiogenesis, fibrosis and coagulation.What the results of this study add? ADAMTS 5, which causes the degradation of the ECM with Aggrecanase activity, was found to be significantly higher in patients diagnosed with cancer and ADAMTS 8 with anti-angiogenesis activity was significantly lower in patients diagnosed with endometrial cancer.What the implications are of these findings for clinical practice and/or further research? In this study, it is understood that the effect of inflammatory mediators is remarkably important in the aetiology of endometrial cancer, as in many types of organ specific cancer.

Ablation of low-molecular-weight FGF2 isoform accelerates murine osteoarthritis while loss of high-molecular-weight FGF2 isoforms offers protection.

FGF2 is an essential growth factor implicated in osteoarthritis (OA), and deletion of full-length FGF2 (Fgf2ALLKO ) leads to murine OA. However, the FGF2 gene encodes both high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms, and the effects of selectively ablating individual isoforms, as opposed to total FGF2, has not been investigated in the context of OA. We undertook this study to examine whether mice lacking HMW FGF2 (Fgf2HMWKO ) or LMW FGF2 (Fgf2LMWKO ) develop OA and to further characterize the observed OA phenotype in Fgf2ALLKO mice. Fgf2HMWKO mice never developed OA, but 6- and 9-month-old Fgf2LMWKO and Fgf2ALLKO mice displayed signs of OA, including eroded articular cartilage, altered subchondral bone and trabecular architecture, and increased OA marker enzyme levels. Even with mechanical induction of OA, Fgf2HMWKO mice were protected against OA, whereas Fgf2LMWKO and Fgf2ALLKO displayed OA-like changes of the subchondral bone. Before exhibiting OA symptoms, Fgf2LMWKO or Fgf2ALLKO joints displayed differential expression of genes encoding key regulatory proteins, including interleukin-1ß, insulin-like growth factor 1, bone morphogenetic protein 4, hypoxia-inducible factor 1, B-cell lymphoma 2, Bcl2-associated X protein, a disintegrin and metalloproteinase with thrombospondin motifs 5, ETS domain-containing protein, and sex-determining region Y box 9. Moreover, Fgf2LMWKO OA cartilage exhibited increased FGF2, FGF23, and FGFR1 expression, whereas Fgf2HMWKO cartilage had increased levels of FGFR3, which promotes anabolism in cartilage. These results demonstrate that loss of LMW FGF2 results in catabolic activity in joint cartilage, whereas absence of HMW FGF2 with only the presence of LMW FGF2 offers protection from OA.

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.