And Metalloproteinase With Thrombospondin Motifs Research Articles

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.

Elevation of MMP1 and ADAMTS5 mRNA expression in glenohumeral synovia of patients with hypercholesterolemia

BackgroundEpidemiological studies have reported a positive association between hypercholesterolemia and shoulder disease. Previous studies have focused on the effect of hypercholesterolemia on tendinopathy. Moreover, hypercholesterolemia has also been linked to joint pathology in the knee and hand. However, the effect of hyperlipidemia on glenohumeral joint remain unclear. A hypercholesterolemic condition has been reported to alter levels of A Disintegrin and Metalloprotease with Thrombospondin Motifs (ADAMTSs) and matrix metalloproteases (MMPs) in synovium of the knee joint. Here, we evaluated the mRNA expression of ADAMTSs and MMPs in the glenohumeral synovium of patients with and without hypercholesterolemia.MethodsStudy participants were 73 patients who underwent arthroscopic rotator cuff repair for degenerative rotator cuff tears. They were divided into two groups according to total cholesterol (TC) and triglyceride levels. Synovial membrane samples were harvested at the rotator interval during surgery, and mRNA expression levels of the aggrecanases ADAM-TS4 and ADAM-TS5 and MMPs (MMP-1, 3, 9, and 13) were analyzed quantitatively.ResultsADAM-TS5 and MMP1 mRNA levels were significantly higher in the high TC group than in the low TC group (P = 0.023 and P = 0.025, respectively). In contrast, no significant differences were observed in ADAMTS4 or MMPs 3, 9, and 13 (ADAMTS4, P = 0.547; MMP3, P = 0.55; MMP9, P = 0.521; and MMP13, P = 0.785).ConclusionHypercholesterolemia may alter MMP1 and ADAMTS5 expression in the synovium of the glenohumeral joint.

IL-33 induces ADAMTS5 expression and cell migration in glioblastoma multiforme.

Glioblastoma multiforme (GBM), characterized by a high rate of proliferation and migration capacity, is an incurable brain tumor in adults. Interleukin-33 (IL-33), a member of the IL-1 cytokine superfamily, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a family of zinc dependent metalloproteinases, are known to have essential roles in GBM migration and invasion. Previous studies have separately revealed elevated expressions of IL-33 and ADAMTS5 in GBM; however, the interaction between IL-33 and ADAMTS5 in GBM remains unclear. Here, using publically available GlioVis and GEPIA programs, we showed that mRNA expressions of IL-33 and ADAMTS5 are significantly high in GBM cells, and a positive correlation between IL-33 and ADAMTS5 was also determined in these cells. In parallel with the mRNA data of IL-33 and ADAMTS5, by Western blot analysis, protein levels were found to be elevated in GBM tissues and increased gradually with the disease progression. Primary GBM cells and low-grade glioma cells were then treated with IL-33 to examine its stimulating effect on ADAMTS5 expression. Exposure to IL-33 raised ADAMTS5 protein levels in a dose-dependent manner. Finally, the wound-healing method was performed to confirm the impact of IL-33 on migration in primary GBM cells. IL-33 promoted migration of primary GBM cells three times higher than untreated GBM cells. Thus, the current study suggests for the first time that IL-33 might have a role in playing a part in GBM progression through induction of ADAMTS5 expression and promotion of migration in GBM cells.

ADAM and ADAMTS disintegrin and metalloproteinases as major factors and molecular targets in vascular malfunction and disease.

A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are two closely related families of proteolytic enzymes. ADAMs are largely membrane-bound enzymes that act as molecular scissors or sheddases of membrane-bound proteins, growth factors, cytokines, receptors and ligands, whereas ADAMTS are mainly secreted enzymes. ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and transmembrane domain. Similarly, ADAMTS family members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but instead of a transmembrane domain they have thrombospondin motifs. Most ADAMs and ADAMTS are activated by pro-protein convertases, and can be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C. Activated ADAMs and ADAMTS participate in numerous vascular processes including angiogenesis, vascular smooth muscle cell proliferation and migration, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs and ADAMTS also play a role in vascular malfunction and cardiovascular diseases such as hypertension, atherosclerosis, coronary artery disease, myocardial infarction, heart failure, peripheral artery disease, and vascular aneurysm. Decreased ADAMTS13 is involved in thrombotic thrombocytopenic purpura and microangiopathies. The activity of ADAMs and ADAMTS can be regulated by endogenous tissue inhibitors of metalloproteinases and other synthetic small molecule inhibitors. ADAMs and ADAMTS can be used as diagnostic biomarkers and molecular targets in cardiovascular disease, and modulators of ADAMs and ADAMTS activity may provide potential new approaches for the management of cardiovascular disorders.

Advances in ADAMTS biomarkers.

A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) are major mediators in extracellular matrix (ECM) turnover and have gained increasing interest over the last years as major players in ECM remodeling during tissue homeostasis and the development of diseases. Although, ADAMTSs are recognized in playing important roles during tissue remodeling, and loss of function in various member of the ADAMTS family could be associated with the development of numerous diseases, limited knowledge is available about their specific substrates and mechanism of action. In this chapter, we will review current knowledge about ADAMTSs and their use as disease biomarkers.

RETRACTED ARTICLE: The protective effects of Olmesartan against interleukin-29 (IL-29)-induced type 2 collagen degradation in human chondrocytes

Statement of Retraction Yunlong Liu, Junyi Liu, Yan Ma, Yongyong Zhang, Qiong Chen, Xin Yang, Yanchun Shang- 2022, The protective effects of Olmesartan against interleukin-29 (IL-29)-induced type 2 collagen degradation in human chondrocytes, Bioengineered 13(1), doi: 10.1080/21655979.2021.1997090 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

The expression of metalloproteinases in the lumbar disc correlates strongly with Pfirrmann MRI grades in lumbar spinal fusion patients

IntroductionIncreased catabolism of the extracellular matrix is observed under degenerative disc disease (DDD). The cleavage of extracellular matrix proteins in the nucleus pulposus (NP) by either matrix metalloproteinases (MMPs) or a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) is believed to be involved in the degeneration, but the mechanisms are not known. Research questionHere, we examine the correlation between expression of several MMPs and ADAMTSs subtypes in lumbar discs from 34 patients with low back pain (LBP) undergoing 1-2 level lumbar fusion surgery (L4/L5 and/or L5/S1) for DDD with or without spondylolisthesis. Materials and MethodsThe mRNA levels of MMPs (subtypes 1, 2, 3, 10, and 13) and ADAMTSs (subtypes 1, 4, and 5) were analyzed using quantitative real-time polymerase chain reaction (RT-qPCR) and correlated to the Pfirrmann magnetic resonance imaging classification system (grade I-V) of lumbar DDD. ResultsWe find a highly significant positive correlation between Pfirrmann grades and the gene expression of MMP1 (r=0.67, p=0.0001), MMP3 (r=0.61, p=0.0002), MMP10 (r=0.6701, p=0.0001), MMP13 (r=0.48, p=0.004), ADAMTS1 (r=0.67, p=0.0001), and ADAMTS5 (r=0.53, p=0.0017). The similar regulation of these transcript suggests their involvement in disc degeneration. Interestingly, a post hoc analysis (uncorrected p-values) also demonstrated a positive correlation between expression of TNF-α, IL-6 and both ADAMTSs/MMPs and the Pfirrmann grades. Discussion and ConclusionThese findings show that disc degradation in DDD is strongly associated with the expression of some metalloproteinases.

Synergistic upregulation of ADAMTS4 (aggrecanase-1) by cytokines and its suppression in knee osteoarthritic synovial fibroblasts

AbstractThe ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-β markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-β, or IL-1α, TNF-α and TGF-β. The signaling molecules' inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-β-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-β-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-β was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-β in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Characterization of novel neuropeptide proteolytic processing involved in dementia pathophysiology

AbstractBackgroundNeuropeptides are neurotransmitter‐like molecules with multiple roles in neuronal activity that are processed into functional peptides (aka proteoforms) by prohormone convertases and other currently unidentified proteases. Both neuropeptides and their proteoforms are implicated in the pathogenesis of different forms of dementia. The neuropeptide VGF is decreased in the cerebrospinal fluid (CSF) and brain of patients with Alzheimer’s disease (AD) and other forms of dementia. VGF356‐375 and VGF1‐40 decrease in the CSF of patients with AD and frontotemporal dementia (FTD), respectively, compared to healthy age‐matched controls. Although many such neuropeptide proteoforms are thought to be involved in dementia, current research has focused on the neuropeptides themselves, leaving the proteoforms and their physiological processing pathways understudied and their contribution to dementia pathogenesis unclear.MethodUsing recombinant proteolysis assays, we aimed to investigate the proteolytic processing of several neuropeptides involved in dementia. Neuropeptides of interest were identified by mass spectrometry analysis of pooled CSF samples obtained from five AD patients and five healthy controls. Proteasix, the MEROPS, and the human protein atlas databases were used to identify novel neuronally‐expressed proteases that may cleave these identified neuropeptides; proteases of interest were selected based on the predicted probability of cleavage. Recombinant protein assays and immunoblots with neuropeptide‐specific antibodies were used to investigate the ability of these proteases to cleave the neuropeptides, and banding patterns were confirmed in triplicate.ResultTwenty‐two neuronally expressed proteases previously linked to dementia were identified that may cleave the neuropeptides chromogranin A (CHGA), secretogranin‐1 (SCG1), secretogranin‐2 (SCG2), secretogranin‐3 (SCG3), and VGF. The proteases a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), calpain‐1 (CAPN1), cathepsin S (CTSS), granzyme A (GZMA), and matrix metalloprotease‐3 (MMP‐3) displayed high probability for neuropeptide proteolysis. These proteases cleaved the neuropeptides into N‐terminal, mid‐region, or C‐terminal neuropeptide proteoforms, and all neuropeptide‐protease combinations except the SCG3‐CAPN1 pair demonstrated cleavage.ConclusionOverall, this work provides mechanistic insight into neuropeptide processing and expands our understanding of neuropeptide proteoform involvement in dementia. Future projects aim to confirm and expand upon these results using mass‐spectrometry, cell culture systems, and CSF and brain protein analysis.