And Metalloproteinase With Thrombospondin Motifs Research Articles

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFβ-1). EVTs contracted in response to TGFβ-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Differential gene expression of ADAMTS-1, ADAMTS-9 and TIMP-3 in periodontitis

ABSTRACT A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are metalloproteinases that bind to components of the extracellular matrix (ECM) to regulate tissue remodeling and homeostasis. ADAMTS can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). Expression of ADAMTS increases under inflammatory conditions. We investigated the mRNA expression of ADAMTS-1, ADAMTS-9 and TIMP-3 genes in both healthy gingival tissues and periodontitis. Clinical periodontal measurements were conducted and gingival biopsies were obtained from stage IIIgrade C generalized periodontitis and healthy (control) groups. mRNA expression was evaluated using real-time quantitative polymerase chain reaction (RTqPCR). All clinical periodontal parameters were significantly higher in the periodontitis group than for the control group. ADAMTS-1 levels were significantly higher in the periodontitis group and were significantly correlated with clinical attachment level and probing pocket depth. Differences in ADAMTS-9 and TIMP-3 mRNA in the periodontitis group compared to the control group were not statistically significant. Increased ADAMTS-1 mRNA expression in periodontitis indicates that members of the ADAMTS family of metalloproteinases are associated with pathogenesis and progression of periodontal disease. Maintaining balance between ADAMTS and TIMP is important for limiting ECM catabolism and preventing tissue damage.

Salinomycin alleviates osteoarthritis progression via inhibiting Wnt/β-catenin signaling

Osteoarthritis (OA) is the most prevalent degenerative whole-joint disease characterized by cartilage degeneration, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis. Currently there are no disease-modifying treatments available for OA because its etiology and pathogenesis are largely unknown. Here we report that a natural carboxylic polyether ionophore that is used as an anti-tumor drug, salinomycin (SAL), may be a promising therapeutic drug for OA in the future. We found that SAL showed no cytotoxicity on mouse chondrocytes and displayed a protective effect against interleukin-1β (IL-1β), in cultured mouse chondrocytes and cartilage explants. Treatment with low SAL concentrations directly upregulated the anabolism factors collagen II and aggrecan, while it inhibited the catabolic factors matrix metalloproteinase-13 (MMP13) and metalloproteinase with thrombospondin motifs-5 (ADAMTS5) to protect against extracellular matrix (ECM) degradation, and also suppressed inflammatory responses in mouse chondrocytes. Furthermore, SAL reduced the severity of OA-associated changes and delayed cartilage destruction, subchondral bone sclerosis, and osteophyte formation in a destabilized medial meniscus (DMM) surgery-induced mouse OA model. Mechanistically, a low SAL concentration induced anabolism and inhibited catabolism in chondrocytes via inhibiting Lrp6 phosphorylation and Wnt/β-catenin signaling. Our results suggested that SAL may serve as a potential disease-modifying therapeutic against OA pathogenesis.

Systemic iron reduction via an iron deficient diet decreases the severity of knee cartilage lesions in the Dunkin-Hartley guinea pig model of osteoarthritis

Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model. Twelve-week-old male Hartley guinea pigs received the standard diet (n=6) or a diet devoid of iron (n=6) for 19-weeks. Iron levels were determined in the serum, liver, and articular cartilage. Knees were collected to assess structural changes related to OA (microcomputed tomography, histopathology). Immunohistochemistry was performed to evaluate the presence and distribution of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ROS-driven 4-hydroxynonenal (4-HNE)-induced protein adducts. Transcript expression was also assessed. Relative to control animals, an iron deficient diet reduced the concentration of this mineral in serum, liver, and articular cartilage. Iron deficient animals had lower histologic OA scores; decreased subchondral bone mineral density was also noted. This reduction in knee joint pathology was accompanied by a decrease in: ADAMTS4 in synovium; and 4-HNE protein adducts from lipid peroxidation in both the menisci and articular cartilage of iron deficient animals. Expression of iron-related genes in these tissues was also altered in treated animals. Results from this study suggest that systemic iron levels may play a role in knee OA pathogenesis, with a short-term deficit in dietary iron reducing the severity of knee cartilage lesions.

Knockdown of Circ_0037658 Alleviates IL-1β-Induced Osteoarthritis Progression by Serving as a Sponge of miR-665 to Regulate ADAMTS5.

Background: Osteoarthritis (OA) is a chronic musculoskeletal degeneration disease which brings great pain to patients and a tremendous burden on the world’s medical resources. Previous reports have indicated that circular RNAs (circRNAs) are involved in the pathogenesis of OA. The purpose of this study was to explore the role and mechanism of circ_0037658 in the OA cell model. Methods: The content of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) was measured using enzyme-linked immunosorbent assay (ELISA). Cell proliferation ability and apoptosis were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EDU), and flow cytometry assays. Western blot assay was used to measure the protein levels of Bcl-2-related X protein (Bax), cleaved-caspase-3, MMP13, Aggrecan, and ADAMTS5. The expression of circ_0037658, microRNA-665 (miR-665), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay were manipulated to analyze the relationships of circ_0037658, miR-665, and ADAMTS5. Results: Human chondrocytes (CHON-001 cells) were treated with interleukin-1β (IL-1β) to establish an OA cell model. Circ_0037658 and ADAMTS5 levels were increased, and miR-665 was decreased in OA cartilage samples and IL-1β-treated chondrocyte cells. Moreover, circ_0037658 silencing promoted proliferation and impaired inflammation, apoptosis, and ECM degradation in IL-1β-treated CHON-001 cells. Mechanically, circ_0037658 acted as a sponge for miR-665 to regulate ADAMTS5 expression. Conclusion: Circ_0037658 knockdown relieved IL-1β-triggered chondrocyte injury via regulating the miR-665/ADAMTS5 axis, promising an underlying therapeutic strategy for OA.

Expression and distribution pattern of aggrecanases and miR-140s in the thickened synovia of shoulder joints in rotator cuff tears: A retrospective observational study.

The rotator cuff (RC) is frequently torn at the enthesis composed of fibrocartilage. We aimed to histopathologically evaluate lining layers and assess the distribution of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, and microRNA (miR)-140s in the synovia of patients with RC tears. We recruited 51 patients who underwent arthroscopic surgical treatment for full-thickness rotator cuff tears, including 26 patients with < 3 cm tear size (group N) and 25 patients with ≥ 3 cm tear size (group W). Biopsied synovia were analyzed using histological and immunohistological techniques for the presence ADAMTS4 and ADAMTS5. The layers of the synovial lining were morphologically classified into 3 grades according to the synovitis score and staining levels of ADAMTSs. The glenohumeral synovia from 8 patients with recurrent shoulder dislocation (group C) were used as controls. Furthermore, in situ hybridization was performed to evaluate the presence of miR-140s in patients with massive tears and recurrent shoulder dislocation. The staining levels were evaluated and analyzed based on comparison between patient groups and correlation between ADAMTS5 and miR-140s. Histological analysis revealed significant differences between groups W and C. ADAMTS5 and ADAMTS4 were strongly expressed in the synovial lining of patients in group W, and this expression was significantly higher than that in groups C and N. In addition, expression of ADAMTS5 was inversely correlated with that of miR-140-3p. This study showed that synovia from group W had a significantly higher rate of severely thickened areas with strong expression of both aggrecanases. Furthermore, the area with weak expression of miR-140-3p showed strong ADAMTS5 expression.

Polydatin inhibits IL-1β-mediated chondrocyte inflammation and ameliorates cartilage degradation: Involvement of the NF-κB and Wnt/β-catenin pathways

Osteoarthritis (OA) is a highly prevalent chronic joint disease that involves extracellular matrix (ECM) degradation and articular cartilage inflammation. Polydatin (PD) can alleviate inflammatory reactions in numerous diseases. The present study aimed to investigate the chondroprotective and anti-inflammatory effects of PD on interleukin (IL)− 1β-treated chondrocytes in vitro and anterior cruciate ligament transection-induced rat OA models in vivo. Primary chondrocytes were isolated from SD rats and cultured. Only second-passage cells were used for subsequent experiments. Counting kit-8, quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, and immunofluorescence were used to detect relevant indices. Rat OA models were established to obtain in vivo data. PD treatment decreased the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and IL-6 during IL-1β-stimulated chondrocyte inflammation. Moreover, PD upregulated aggrecan and collagen II expression, whereas downregulated a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinase-13 (MMP-13) expression on IL-1β-mediated chondrocytes. Additionally, PD reduced IL-1β-stimulated NF-κB and Wnt/β-catenin activation and nuclear translocation. The results of histological analysis and scoring revealed that OA in the rat models was effectively ameliorated by the intra-articular injection of PD. PD suppressed IL-1β-stimulated iNOS, COX-2, NO, and PGE2 production, TNF-α, IL-6, collagen X, MMP-13, and ADAMTS-5 expression, collagen II and aggrecan degeneration by inhibiting NF-κB and Wnt/β-catenin signaling in vitro. PD also mitigated OA progression in the rat models, thereby providing reliable data that PD could serve as a promising candidate for OA therapy.

Cepharanthine Ameliorates Chondrocytic Inflammation and Osteoarthritis via Regulating the MAPK/NF-κB-Autophagy Pathway.

Osteoarthritis is a worldwide joint disease caused by abnormal chondrocytic metabolism. However, traditional therapeutic methods aimed at anti-inflammation for early-stage disease are palliative. In the present study, we demonstrated that cepharanthine (CEP), extracted from the plant Stephania cepharantha, exerted protective medicinal efficacy on osteoarthritis for the first time. In our in vitro study, CEP suppressed the elevated expression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and inducible nitric oxide synthase (iNOS) stimulated by IL-1β or TNF-α by inhibiting the activation of MAPK and NF-κB signaling pathways, and upregulated the protein expression of aggrecan, collagen II, and Sox9. Also, CEP could reverse the reduced level of cellular autophagy in IL-1β or TNF-α–induced chondrocytes, indicating that the protective effect of CEP on osteoarthritis was achieved by restoring MAPK/NF-κB-mediated autophagy. Furthermore, in a murine OA model, CEP mitigated cartilage degradation and prevented osteoarthritis in the CEP-treated groups versus the OA group. Hence, our results revealed the therapeutic prospect of CEP for anti-osteoarthritic treatment.

Selection and characterization of DNA aptamers inhibiting a druggable target of osteoarthritis, ADAMTS-5

There is a critical need for the development of more potent inhibitors for osteoarthritis (OA) therapy given the poor life quality of arthritis patients. Aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is an established drug target identified for osteoarthritis. In this study, we evolved and characterized two new DNA aptamer inhibitors of ADAMTS-5, namely apt21 and apt25. The aptamers exhibited nanomolar binding affinity and high specificity against ADAMTS-5. KD values of apt21 and apt25 were determined by the Enzyme-linked Oligonucleotide Assay (ELONA) at 1.54 ± 0.16 nM and 1.79 ± 0.08 nM, respectively. Circular Dichroism (CD) analysis demonstrated that both aptamers formed monovalent cation dependent G-quadruplex structures. Calcium ions did not affect the binding of the aptamers to ADAMTS-5. The inhibitory effects of apt21 and apt25 on ADAMTS-5 were evaluated by the Förster Resonance Energy Transfer (FRET) assay, in which IC50 values of apt21 and apt25 were estimated at 52.76 ± 6.70 μM and 61.14 ± 9.67 μM, respectively. These two aptamers are the first DNA G-quadruplex aptamers demonstrated to inhibit ADAMTS-5 and could have value for OA therapy.

ADAMTS4-specific MR probe to assess aortic aneurysms in vivo using synthetic peptide libraries

The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it’s potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture.

ADAMTS-4 as a possible distinguishing indicator between osteoarthritis and haemophilic arthropathy.

IntroductionOsteoarthritis (OA) and haemophilic arthropathy (HA) are clinically similar, but pathologically distinct conditions which result in joint pain and loss of function. Distinguishing their disease mechanisms is therefore a key step in the development of curative therapy, as opposed to current symptomatic treatments. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 is a metzincin‐family member proteoglycan with known local involvement in OA pathogenesis.AimTo investigate the potential differences and discriminatory potential of ADAMTS‐4 between OA and HA patients.MethodsWe determined ADAMTS‐4 plasma concentrations by ELISA in patients with HA and OA. This pilot cross‐sectional study included N = 40 male participants equally divided across four subgroups: haemophilia patients with severe or mild HA and control subjects with severe or mild/no OA.ResultsOur study showed a striking elevation in plasma ADAMTS‐4 expression levels in HA patients as compared to OA, as well as an increase in patients with severe as compared to mild HA. By performing the binomial logistical analysis and fitting the receiver–operator curve (ROC) (cut‐off probability .5), ADAMTS‐4 had a sensitivity of 95% and specificity of 50% in discriminating between HA and OA among our study participants.ConclusionUncovering the marked differences in plasma levels of ADAMTS‐4 in patients with HA versus OA potentially sheds new light on the mechanisms of HA pathogenesis and could foster more research into the roles ADAMTS‐4 and other matrix metalloproteinases (MMPs) play in HA versus OA.

Zoledronate promotes ECM degradation and apoptosis via Wnt/β-catenin.

This study examined the potential mechanism of zoledronate on interleukin (IL)-1β-induced temporomandibular joint osteoarthritis (TMJOA) chondrocytes, using IL-1β-induced rabbit immortalized mandibular condylar chondrocytes cultured with zoledronate. Cell viability, apoptosis, mRNA, and protein expression of relevant genes involved in extracellular matrix (ECM) degradation, apoptosis, and Wnt/β-catenin signaling were examined. The involvement of the Wnt/β-catenin signaling was examined using Wnt/β-catenin inhibitor (2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol (XAV-939)) and activator lithium chloride (LiCl). Aggrecan and type II collagen were downregulated by zoledronate, especially with 100 nM for 48 h (p < 0.01), consistently with the upregulation of A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) (p < 0.001), matrix metalloprotease-9 (MMP-9) (p < 0.01), caspase-3 (p < 0.001) and downregulation of proliferating cell nuclear antigen (PCNA) (p < 0.01). The apoptotic rate increased from 34.1% to 45.7% with 100 nM zoledronate for 48 h (p < 0.01). The effects of zoledronate on ADAMTs4 (p < 0.001), MMP-9 (p < 0.001), caspase-3 (p < 0.001), and PCNA (p < 0.01) were reversed by XAV-939, while LiCl increased caspase-3 expression (p < 0.01). In conclusion, zoledronate enhances IL-1β-induced ECM degradation and cell apoptosis in TMJOA chondrocytes. Wnt/β-catenin signaling might be involved in this process, but additional studies are necessary to determine the exact involvement of Wnt/β-catenin signaling in chondrocytes after zoledronate treatment.

The Effects of TNF-α Inhibition on the Metabolism of Cartilage: Relationship between KS, HA, HAPLN1 and ADAMTS4, ADAMTS5, TOS and TGF-β1 Plasma Concentrations in Patients with Juvenile Idiopathic Arthritis.

We assessed the effect of 24-month anti-tumor necrosis factor alpha (TNF-α) treatment on the remodeling of the cartilage extracellular matrix (ECM) in patients with juvenile idiopathic arthritis (JIA). Methods: Quantitative evaluation of keratan sulfate (KS), hyaluronic acid (HA), hyaluronan and proteoglycan link protein 1 (HAPLN1), as potential biomarkers of joint dysfunction, and the levels of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and 5, total oxidative status (TOS) and transforming growth factor (TGF-β1) was performed (using immunoenzymatic methods) in blood obtained from patients before and after 24 months of etanercept (ETA) treatment. Results: When compared to the controls, KS, HA and HAPLN1 levels were significantly higher in patients with an aggressive course of JIA qualified for ETA treatment. An anti-cytokine therapy leading to clinical improvement promotes the normalization only of the HA level. Proteolytic and pro-oxidative factors, present in high concentrations in patients before the treatment, correlated with HAPLN1, but not with KS and HA levels. In these patients, negative correlations were found between the levels of TGF-β1 and KS, HA and HAPLN1. Conclusion: The anti-TNF-α therapy used in patients with JIA has a beneficial effect on ECM cartilage metabolism, but it does not completely regenerate it. The changes in the plasma HA level during the anti-cytokine therapy suggest its potential diagnostic utility in monitoring of disease activity and may be used to assess the efficacy of ETA treatment.

IMPACT OF 45S5-BIOACTIVE GLASS ON SYNOVIAL CELLS IN KNEE OSTEOARTHRITIS - IN VITRO STUDY

Purpose: In osteoarthritis (OA) inflammation is considered a crucial factor for cartilage destruction and disease progression. Metalloproteinases, matrix metalloproteinases (MMPs) and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS), play a key role in the inflammation process in OA through their chondrodestructive potential. Further understanding of OA inflammation mechanisms, especially considering MMPs and ADAMTS, holds the opportunity for future treatment options. 45S5-bioactive glass (BG) has traditionally been used as a bone substitute material, but its bioactive characteristics have sparked a wide discussion for more widespread applications.

BIOLOGICAL VARIATION OF THE HUMAN AGGRECAN ARGS NEOEPITOPE IN SYNOVIAL FLUID AND SERUM IN EARLY-STAGE KNEE OSTEOARTHRITIS

Purpose: In the ongoing search for a successful disease-modifying osteoarthritis drug (DMOAD), a common approach is inhibition of the aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) which has been identified as the key protease in pathological degradation of human aggrecan. To monitor aggrecanase activity, and inhibition thereof, quantification of the aggrecanase generated aggrecan ARGS neoepitope has been used. We here describe the biological variation of the ARGS neoepitope in knee joint fluid and serum between and within individuals with early-stage knee OA, a target population for DMOADs.

Taurine attenuates ER stress‑associated apoptosis and catabolism in nucleus pulposus cells.

Nucleus pulposus (NP) apoptosis and subsequent excessive degradation of the extracellular matrix (ECM) are key pathological characteristics of intervertebral disc degeneration (IDD). The present study aims to examine the signaling processes underlying the effects of taurine on IDD, with specific focus on endoplasmic reticulum (ER) stress-mediated apoptosis and ECM degradation, in NP cells. To clarify the role of taurine in IDD, NP cells were treated with various concentrations of taurine and IL-1β or thapsigargin (TG). Cell Counting Kit-8, western blotting, TUNEL, immunofluorescence assays and reverse transcription-quantitative PCR were applied to measure cell viability, the expression of ER stress-associated proteins (GRP78, CHOP and caspase-12), apoptosis and the levels of metabolic factors associated with ECM (MMP-1, 3, 9, ADAMTS-4, 5 and collagen II), respectively. Taurine was found to attenuate ER stress and prevent apoptosis in NP cells induced by IL-1β treatment. Additionally, taurine significantly decreased the expression of ER stress-activated glucose regulatory protein 78, C/EBP homologous protein and caspase-12. TUNEL results revealed that taurine decreased the number of apoptotic TG-treated NP cells. TG-treated NP cells also exhibited characteristics of increased ECM degradation, supported by observations of increased MMP-1, MMP-3, MMP-9 and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 expression in addition to decreased collagen-II expression. However, taurine treatment significantly reversed all indicators of excessive ECM catabolism aforementioned. These data suggest that taurine can mediate protection against apoptosis and ECM degradation in NP cells by inhibiting ER stress, implicating therapeutic potential for the treatment of IDD.

Cleavage of LOXL1 by BMP1 and ADAMTS14 Proteases Suggests a Role for Proteolytic Processing in the Regulation of LOXL1 Function.

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.

Association between primary osteoarthritis and ADAMTS14 single nucleotide polymorphism in Egyptian population: a case-control study

BackgroundPrimary osteoarthritis is considered one of the most common and the most studied musculoskeletal disorder. Nevertheless, the risk factors remain unclear. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 14 (ADAMTS14) gene is involved in the cleavage of amino-terminal propeptides from type II procollagen, a necessary step in the formation of collagen fibers. The abnormal metabolism of collagen fibers type II leads to a decreased mechanical strength of joint cartilage which is one of the most important contributing factors to joint osteoarthritis. We aimed at investigating the association between primary osteoarthritis and ADAMTS14 gene rs4747096 single nucleotide polymorphism in a sample of Egyptian patients and analyzing the relationship between this genetic polymorphism with the severity of osteoarthritis. Sixty-five Egyptian patients who fulfilled the American College of Rheumatology criteria for primary knee osteoarthritis were compared with thirty-one apparently healthy subjects. Genotyping was performed by TaqMan single nucleotide polymorphism genotyping assay.ResultsThere was a statistically significantly higher frequency of AA genotype among osteoarthritis patients compared to the control group (P = 0.004). The number of affected hand joints was significantly higher among patients with ADAMTS14 AA genotype in comparison to patients with ADAMTS14 AG genotype (P = 0.002). In addition, AA genotype was associated with statistically significantly higher Kellgren-Lawrence radiological grades in the knee and hand joints (proximal interphalangeal and thumb interphalangeal joints) (P = 0.037, 0.003, and 0.030 respectively).ConclusionThe study showed an association between the AA genotype of ADAMTS14 gene rs4747096 single nucleotide polymorphism with knee and hand osteoarthritis and osteoarthritis severity in these joints. The AA genotype of ADAMTS14 gene rs4747096 single nucleotide polymorphism could be implicated in the increased incidence of primary osteoarthritis development and elevated disease severity among the Egyptian population.

Circular RNA circ_0020014 contributes to osteoarthritis progression via miR-613/ADAMTS5 axis.

Circular RNAs have been shown to be significant regulators in osteoarthritis (OA), whereas the functional effect of circ_0020014 in OA remains unclear. Our goal was to try and understand the underlying regulatory mechanism of circ_0020014 in OA. The cartilage tissue was obtained from OA patients and trauma patients. Interleukin-1β (IL-1β)-treated chondrocytes (CHON-001) were used as the in vitro cellular model for OA. The expression levels of circ_0020014, microRNA-613 (miR-613), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were examined by real-time quantitative polymerase chain reaction. The protein level was detected using the Western blot assay. Cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide and flow cytometry assays, respectively. The secretion of inflammatory cytokine was determined by enzyme-linked immunosorbent assay. Circ_0020014 was upregulated in OA cartilage tissues and IL-1β-treated CHON-001 cells, compared with that in healthy cartilage tissues and untreated cells. IL-1β treatment induced cell injury by promoting inflammation and apoptosis, and inhibiting cell viability and extracellular matrix accumulation in chondrocytes. Circ_0020014 knockdown significantly protected CHON-001 cells from IL-1β-induced cell dysfunction. MiR-613 was targeted by circ_0020014 and negatively regulated ADAMTS5 expression. In addition, miR-613 downregulation or ADAMTS5 overexpression partly lessened the protective effect of circ_0020014 knockdown on IL-1β-treated CHON-001 cells. Collectively, circ_0020014 acted as a miR-613 sponge to regulate ADAMTS5 expression, thereby protecting chondrocytes from IL-1β-induced inflammatory damage, which might be a novel diagnostic marker for OA.

Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study.

Osteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1β-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1β-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints.