And Metalloproteinase With Thrombospondin Motifs Research Articles

HTRA1 from OVX rat osteoclasts causes detrimental effects on endplate chondrocytes through NF-κB

Endplate osteochondritis is considered one of the major causes of intervertebral disc degeneration (IVDD) and low back pain. Menopausal women have a higher rate of endplate cartilage degeneration than similarly aged men, but the related mechanisms are still unclear. Subchondral bone changes, mainly mediated by osteoblasts and osteoclasts, are considered an important reason for the degeneration of cartilage. This work explored the role of osteoclasts in endplate cartilage degeneration, as well as its underlying mechanisms. A rat ovariectomy (OVX) model was used to induce estrogen deficiency. Our experiments indicated that OVX significantly promoted osteoclastogenesis and anabolism and catabolism changes in endplate chondrocytes. OVX osteoclasts cause an imbalance between anabolism and catabolism in endplate chondrocytes, as shown by a decrease in anabolic markers such as Aggrecan and Collagen II, and an increase in catabolic markers such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinases (MMP13). Osteoclasts were also confirmed in this study to be able to secrete HtrA serine peptidase 1 (HTRA1), which resulted in increased catabolism in endplate chondrocytes through the NF-κB pathway under estrogen deficiency. This study demonstrated the involvement and mechanism of osteoclasts in the anabolism and catabolism changes of endplate cartilage under estrogen deficiency, and proposed a new strategy for the treatment of endplate osteochondritis and IVDD by targeting HTRA1.

ADAMTS Proteases: Importance in Animal Reproduction.

Many reproductive physiological processes, such as folliculogenesis, ovulation, implantation, and fertilization, require the synthesis, remodeling, and degradation of the extracellular matrix (ECM). The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family genes code for key metalloproteinases in the remodeling process of different ECM. Several genes of this family encode for proteins with important functions in reproductive processes; in particular, ADAMTS1, 4, 5 and 9 are genes that are differentially expressed in cell types and the physiological stages of reproductive tissues. ADAMTS enzymes degrade proteoglycans in the ECM of the follicles so that the oocytes can be released and regulate follicle development during folliculogenesis, favoring the action of essential growth factors, such as FGF-2, FGF-7 and GDF-9. The transcriptional regulation of ADAMTS1 and 9 in preovulatory follicles occurs because of the gonadotropin surge in preovulatory follicles, via the progesterone/progesterone receptor complex. In addition, in the case of ADAMTS1, pathways involving protein kinase A (PKA), extracellular signal regulated protein kinase (ERK1/2) and the epidermal growth factor receptor (EGFR) might contribute to ECM regulation. Different Omic studies indicate the importance of genes of the ADAMTS family from a reproductive aspect. ADAMTS genes could serve as biomarkers for genetic improvement and contribute to enhance fertility and animal reproduction; however, more research related to these genes, the synthesis of proteins encoded by these genes, and regulation in farm animals is needed.

The Comprehensive Analysis of Specific Proteins as Novel Biomarkers Involved in the Diagnosis and Progression of Gastric Cancer.

Gastric cancer (GC) cases are predicted to rise by 2040 to approximately 1.8 million cases, while GC-caused deaths to 1.3 million yearly worldwide. To change this prognosis, there is a need to improve the diagnosis of GC patients because this deadly malignancy is usually detected at an advanced stage. Therefore, new biomarkers of early GC are sorely needed. In the present paper, we summarized and referred to a number of original pieces of research concerning the clinical significance of specific proteins as potential biomarkers for GC in comparison to well-established tumor markers for this malignancy. It has been proved that selected chemokines and their specific receptors, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), specific proteins such as interleukin 6 (IL-6) and C-reactive protein (CRP), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), as well as DNA- and RNA-based biomarkers, and c-MET (tyrosine-protein kinase Met) play a role in the pathogenesis of GC. Based on the recent scientific literature, our review indicates that presented specific proteins are potential biomarkers in the diagnosis and progression of GC as well as might be used as prognostic factors of GC patients' survival.

CHANGES TO LEVELS OF CHONDROCYTE-ASSOCIATED ADAMTS-4 IN EARLY HUMAN OSTEOARTHRITIS

Early changes within articular cartilage during human idiopathic osteoarthritis are poorly understood. However alterations to chondrocyte morphology occur with the development of fine cytoplasmic processes and cell clusters, potentially playing a role in cartilage degeneration. The aggrecanase ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs-4) has been implicated as an important factor in cartilage degradation, so we investigated the relationship between chondrocyte morphology and levels of ADAMTS-4 in both non-degenerate and mildly osteoarthritic human cartilage.Human femoral heads were obtained following consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants of normal (grade 0; G0) and mildly osteoarthritic (grade 1; G1) cartilage were labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate). Explants were cryosectioned (30μm sections), and labelled for ADAMTS-4 by fluorescence immunohistochemistry. Sections were imaged with confocal microscopy, allowing the semi-quantitative analysis of ADAMTS-4 and 3D visualisation of in situ cell morphology.With cartilage degeneration from G0 to G1, there was a decrease in the proportion of chondrocytes with normal rounded morphology (P<0.001) but an increase in the proportion of cells with processes (P<0.01) and those in clusters (P<0.001;[4(1653)]; femoral heads:cells). Although average levels of ADAMTS-4 for all cells was the same between G0 and G1 (P>0.05), a change was evident in the distribution curves for cell-specific ADAMTS-4 labelling. Cell-by-cell analysis showed that ADAMTS-4 levels were higher in chondrocytes with cytoplasmic processes compared to normal cells (P=0.044) however cells in clusters had lower levels than normal cells (P=0.003;[3(436)]). Preliminary data suggested that ADAMTS-4 levels increased with larger chondrocyte clusters.These results suggest complex heterogeneous changes to levels of cell-associated ADAMTS-4 with early cartilage degeneration – increasing in cells with processes and initially decreasing in clusters. Increased levels of ADAMTS-4 are likely to produce focal areas of matrix weakness potentially leading to early cartilage degeneration.

IRISIN STIMULATES ANABOLISM AND MATRIX SYNTHESIS IN HUMAN NUCLEUS PULPOSUS CELLS IN VITRO: NEW INSIGHTS INTO A CROSS-TALK BETWEEN THE MUSCLE AND THE INTERVERTEBRAL DISC

This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.

Load-induced blood marker kinetics in patients with medial knee compartment osteoarthritis are associated with accumulated load and patient reported outcome measures

Background: This study aimed to quantify the mechanoresponse of 10 blood marker candidates for joint metabolism to a walking stress test in patients with knee osteoarthritis and to determine the association among marker kinetics and with accumulated load and patient reported outcomes. Methods: 24 patients with knee osteoarthritis completed questionnaires, and a walking stress test with six blood serum samples and gait analysis. Concentrations of cartilage oligomeric matrix protein (COMP), matrix metalloproteinases (MMP)-1, -3, and -9, epitope resulting from cleavage of type II collagen by collagenases (C2C), type II procollagen (CPII), interleukin (IL)-6, proteoglycan (PRG)-4, A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and resistin were determined by enzyme-linked immunosorbent assays, Joint load (moments and compartmental forces) was estimated using musculoskeletal modeling using gait analysis data. Results: COMP and MMP-3 showed an immediate increase after the walking stress followed by a decrease. MMP-9 and resistin showed a delayed decrease below pre-stress levels. ∆COMP correlated with ∆MMP-3 for most time points. ∆MMP-9 correlated with ∆resistin for most time points. The load-induced increase in blood marker levels correlated among blood markers and time points. C2C and resistin correlated positively and C2C/CPII and MMP2 correlated negatively with load during gait. Immediate relative ∆CPII and ∆MMP1 and delayed relative ∆COMP, ∆IL6, ∆C2C, ∆CPII, ∆MMP1 and ∆MMP3 correlated with the load accumulated during the walking stress. Baseline C2C levels correlated with Knee Osteoarthritis Outcome Score (KOOS) subscales and load-induced changes in MMP-3 with KOOS and Short Form 36 quality of life subscores (P<0.05). Conclusions: The distinct and differentiated physiological response to the walking stress depends on accumulated load and appears relevant for patient reported osteoarthritis outcome and quality of life and warrants further investigation in the context of disease progression. ClinicalTrials.gov registration: NCT02622204

Changes in the Extracellular Matrix in Endometrial and Cervical Cancer: A Systematic Review

Endometrial and cervical cancers are the two most common gynaecological malignancies and among the leading causes of death worldwide. The extracellular matrix (ECM) is an important component of the cellular microenvironment and plays an important role in developing and regulating normal tissues and homeostasis. The pathological dynamics of the ECM contribute to several different processes such as endometriosis, infertility, cancer, and metastasis. Identifying changes in components of ECM is crucial for understanding the mechanisms of cancer development and its progression. We performed a systematic analysis of publications on the topic of changes in the extracellular matrix in cervical and endometrial cancer. The findings of this systematic review show that matrix metalloproteinases (MMP) play an important role impacting tumour growth in both types of cancer. MMPs degrade various specific substrates (collagen, elastin, fibronectin, aggrecan, fibulin, laminin, tenascin, vitronectin, versican, nidogen) and play a crucial role in the basal membrane degradation and ECM components. Similar types of MMPs were found to be increased in both cancers, namely, MMP-1, MMP-2, MMP-9, and MMP-11. Elevated concentrations of MMP-2 and MMP-9 were correlated with the FIGO stage and are associated with poor prognosis in endometrial cancer, whereas in cervical cancer, elevated concentrations of MMP-9 have been associated with a better outcome. Elevated ADAMTS levels were found in cervical cancer tissues. Elevated disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) levels were also found in endometrial cancer, but their role is still unclear. Following these findings, this review reports on tissue inhibitors of ECM enzymes, MMPs, and ADAMTS. The present review demonstrates changes in the extracellular matrix in cervical and endometrial cancers and compared their effect on cancer development, progression, and patient prognosis.

Cyasterone inhibits IL-1β-mediated apoptosis and inflammation via the NF-κB and MAPK signaling pathways in rat chondrocytes and ameliorates osteoarthritisin vivo.

Osteoarthritis is a prevalent global joint disease, which is characterized by inflammatory reaction and cartilage degradation. Cyasterone, a sterone derived from the roots of Cyathula officinalis Kuan, exerts protective effect against several inflammation-related diseases. However, its effect on osteoarthritis remains unclear. The current study was designed to investigate the potential anti-osteoarthritis activity of cyasterone. Primary chondrocytes isolated from rats induced by interleukin (IL)-1β and a rat model stimulated by monosodium iodoacetate (MIA) were used for in vitro and in vivo experiments, respectively. The results of in vitro experiments showed that cyasterone apparently counteracted chondrocyte apoptosis, increased the expression of collagen II and aggrecan, and restrained the production of the inflammatory factors inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), metalloproteinase-3 (MMP-3), and metalloproteinase-13 (MMP-13) induced by IL-1β in chondrocytes. Furthermore, cyasterone ameliorated the inflammation and degenerative progression of osteoarthritis potentially by regulating the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. For in vivo experiments, cyasterone significantly alleviated the inflammatory response and cartilage destruction of rats induced by monosodium iodoacetate, where dexamethasone was used as the positive control. Overall, this study laid a theoretical foundation for developing cyasterone as an effective agent for the alleviation of osteoarthritis.

Kallistatin attenuates inflammatory response in rheumatoid arthritis via the NF-κB signaling pathway

Cartilage degeneration and inflammation are important features of rheumatoid arthritis (RA). Chondrocyte inflammation and apoptosis have been increasingly demonstrated to be related to cartilage decomposition. In this study, we analyzed the protective role of kallistatin against RA and its associated mechanisms. We obtained in vitro and in vivo RA models using IL-1β and heat-inactivated Mycobacterium tuberculosis, respectively. Our results showed that kallistatin mitigated IL-1β-mediated chondrocyte apoptosis and inhibited the synthesis of ECM-degrading generation, like matrix metalloproteinase (MMP)-3/13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4/5, in IL-1β-mediated chondrocytes. Furthermore, kallistatin markedly suppressed IL-1β-mediated inflammation while decreasing the levels of inflammatory factors and mediators via the NF-κB pathway. Daily administration of kallistatin reduced the expression levels of PGE2, TNF-α, IL-1β, and IL-6. Histochemical analysis revealed that the kallistatin-treated rats exhibited reduced RA severity compared with control mice. In summary, kallistatin suppressed IL-1β-mediated inflammation in chondrocytes via the NF-κB pathway. Administration of kallistatin remarkably inhibited RA development, accompanied by reduced inflammation and apoptosis. Therefore, kallistatin administration can be used as a candidate therapeutic strategy for RA.

Hydroxytyrosol Ameliorates Intervertebral Disc Degeneration and Neuropathic Pain by Reducing Oxidative Stress and Inflammation.

Low back pain (LBP) seriously affects human quality of life. Intervertebral disc degeneration (IVDD) is the main pathological factor that leads to LBP, but the pathological mechanism underlying IVDD has not been fully elucidated. Neuropathic pain caused by IVDD is an important pathological factor affecting people's daily lives. Therefore, it is very important to identify therapeutic drugs to ameliorate IVDD and secondary neuropathic pain. Hydroxytyrosol (HT) is a natural compound derived from olive leaves and oil and has anti-inflammatory, antioxidant, and antitumor activities and other properties. In this study, TNF-α-stimulated human nucleus pulposus cells (HNPCs) were used to simulate the local inflammatory microenvironment observed in IVDD in vitro to explore the role of HT in alleviating various pathological processes associated with IVDD. A rat needle puncture model was used to further explore the role of HT in alleviating IVDD. Lipopolysaccharide (LPS) was used to stimulate microglia in vitro to comprehensively explore the role of HT in alleviating neuropathic pain, and a rat model involving chronic compression of the dorsal root ganglion (CCD) was established to simulate the neuropathic pain caused by IVDD. This study suggests that HT reduces the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and matrix metalloproteinase-13 (MMP-13); inhibits the production of mitochondrial reactive oxygen species (ROS); and maintains mitochondrial homeostasis. Thus, HT appears to reduce the rate of apoptosis and mitigate the loss of major intervertebral disc components by inhibiting the nuclear factor kappa-B (NF-κB) signaling pathway. Moreover, HT inhibited the secretion of COX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and iNOS and activation of the NLRP3 inflammasome in microglia by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and extracellular regulated protein kinase (ERK) signaling pathways. In conclusion, HT plays a protective role against IVDD and secondary neuropathic pain by inhibiting the NF-κB, PI3K/AKT, and ERK signaling pathways.

Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition.

Osteoarthritis (OA) is the most common degenerative joint disease. In 1999, two members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family of metalloproteinases, ADAMTS4 and ADAMTS5, or aggrecanases, were identified as the enzymes responsible for aggrecan degradation in cartilage. The first aggrecanase inhibitors targeted the active site by chelation of the catalytic zinc ion. Due to the generally disappointing performance of zinc-chelating inhibitors in preclinical and clinical studies, inhibition strategies tried to move away from the active-site zinc in order to improve selectivity. Exosite inhibitors bind to proteoglycan-binding residues present on the aggrecanase ancillary domains (called exosites). While exosite inhibitors are generally more selective than zinc-chelating inhibitors, they are still far from fulfilling their potential, partly due to a lack of structural and functional data on aggrecanase exosites. Filling this gap will inform the design of novel potent, selective aggrecanase inhibitors.

Protective effects of equol on the cartilage and subchondral bone in ovariectomized rats with osteoarthritis.

Objective(s):This study aimed to determine the therapeutic effect of equol (EQ) on osteoporotic osteoarthritis (OP OA). Materials and Methods:Thirty-six 12-week-old female Sprague-Dawley rats were randomly divided into sham group, OP OA group, and EQ group (n=12). OP OA was induced by anterior cruciate ligament transection (ACLT) combined with ovariectomy (OVX). EQ was orally administrated (10 μg/g/day) after the operation for 12 weeks. The efficacy was evaluated by gross pathology and histopathologic evaluation. The underlying mechanism was investigated by immunohistochemical analysis, micro-computed tomography (micro-CT) scanning, and tartrate-resistant acid phosphatase (TRAP) staining. Results:EQ effectively retarded cartilage degeneration, decreased the levels of matrix metalloproteinases-13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), nuclear factor-kappa B P65 (NF-κB P65) and caspase-3, and increased the levels of collagen type II (Col-II), Col-I, aggrecan (AGG), and inhibitor of NF-κB α (IκBα) in the cartilage. In addition, EQ increased bone mineral density, improved the microstructural parameters of the subchondral bone (SB), and decreased the number of osteoclasts. Conclusion:EQ exerted a chondroprotective effect on OP OA in rats, associated with inhibition of the NF-κB signaling pathway and chondrocyte apoptosis. Furthermore, EQ showed an osteoprotective effect on SB via inhibiting osteoclastic activities.

Metzincin metalloproteases in PGC migration and gonadal sex conversion

Development of a functional gonad includes migration of primordial germ cells (PGCs), differentiations of somatic and germ cells, formation of primary follicles or spermatogenic cysts with somatic gonadal cells, development and maturation of gametes, and subsequent releasing of mature germ cells. These processes require extensive cellular and tissue remodeling, as well as broad alterations of the surrounding extracellular matrix (ECM). Metalloproteases, including MMPs (matrix metalloproteases), ADAMs (a disintegrin and metalloproteinases), and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), are suggested to have critical roles in the remodeling of the ECM during gonad development. However, few research articles and reviews are available on the functions and mechanisms of metalloproteases in remodeling gonadal ECM, gonadal development, or gonadal differentiation. Moreover, most studies focused on the roles of transcription and growth factors in early gonad development and primary sex determination, leaving a significant knowledge gap on how differentially expressed metalloproteases exert effects on the ECM, cell migration, development, and survival of germ cells during the development and differentiation of ovaries or testes. We will review gonad development with focus on the evidence of metalloprotease involvements, and with an emphasis on zebrafish as a model for studying gonadal sex differentiation and metalloprotease functions.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

In vitro study. To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. hNPCs were exposed to 5, 10, and 25ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1β gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10ng/mL IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with irisin). Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1β mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1β levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1β and ADAMTS-5 levels. Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

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.