Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Evaluating anticancer activity of emodin by enhancing antioxidant activities and affecting PKC/ADAMTS4 pathway in thioacetamide-induced hepatocellular carcinoma in rats.

Emodin is a naturally occurring anthraquinone derivative with a wide range of pharmacological activities, including neuroprotective and anti-inflammatory activities. We aim to assess the anticancer activity of emodin against hepatocellular carcinoma (HCC) in rat models using the proliferation, invasion, and angiogenesis biomarkers. After induction of HCC, assessment of the liver impairment and the histopathology of liver sections were investigated. Hepatic expression of both mRNA and protein of the oxidative stress biomarkers, HO-1, Nrf2; the mitogenic activation biomarkers, ERK5, PKCδ; the tissue destruction biomarker, ADAMTS4; the tissue homeostasis biomarker, aggregan; the cellular fibrinolytic biomarker, MMP3; and of the cellular angiogenesis biomarker, VEGF were measured. Emodin increased the survival percentage and reduced the number of hepatic nodules compared to the HCC group. Besides, emodin reduced the elevated expression of both mRNA and proteins of all PKC, ERK5, ADAMTS4, MMP3, and VEGF compared with the HCC group. On the other hand, emodin increased the expression of mRNA and proteins of Nrf2, HO-1, and aggrecan compared with the HCC group. Therefore, emodin is a promising anticancer agent against HCC preventing the cancer prognosis and infiltration. It works through many mechanisms of action, such as blocking oxidative stress, proliferation, invasion, and angiogenesis.

ADAMTS4 is a crucial proteolytic enzyme for versican cleavage in the amnion at parturition.

Hyalectan cleavage may play an important role in extracellular matrix remodeling. However, the proteolytic enzyme responsible for hyalectan degradation for fetal membrane rupture at parturition remains unknown. Here, we reveal that versican (VCAN) is the major hyalectan in the amnion, where its cleavage increases at parturition with spontaneous rupture of membrane. We further reveal that ADAMTS4 is a crucial proteolytic enzyme for VCAN cleavage in the amnion. Inflammatory factors may enhance VCAN cleavage by inducing ADAMTS4 expression and inhibiting ADAMTS4 endocytosis in amnion fibroblasts. In turn, versikine, the VCAN cleavage product, induces inflammatory factors in amnion fibroblasts, thereby forming a feedforward loop between inflammation and VCAN degradation. Mouse studies show that intra-amniotic injection of ADAMTS4 induces preterm birth along with increased VCAN degradation and proinflammatory factors abundance in the fetal membranes. Conclusively, there is enhanced VCAN cleavage by ADAMTS4 in the amnion at parturition, which can be reenforced by inflammation.

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

Oxidized LDL-mediated upregulation of ADAMTS-4 in monocytes/macrophages involves ROS-NF-κB-SIRT-1 pathway.

Background and aims: ADAMTS-4 is a protease enzyme involved in vascular remodeling and atherosclerosis. It was found to be upregulated in macrophages seen in atherosclerotic lesions. This study aimed to investigate the expression and regulation of ADAMTS-4 in oxidized LDL-induced human monocytes/macrophages system. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from human blood, and treated with oxidized LDL (50 µg mL-1) were used as the model system for the study. mRNA and protein expressions were studied by PCR, ELISA, and western blot analysis. ROS production and cell viability were determined by DCFDA staining and MTT assay, respectively. Results: In the presence of oxidized LDL, monocytes get differentiated into macrophages, which were confirmed by the increased expression of macrophage differentiation markers and pro-inflammatory cytokine TNF-α. Oxidized LDL increased the mRNA and protein expression of ADAMTS-4 in monocytes/macrophages. N- Acetyl cysteine, ROS scavenger, downregulate the protein expression of ADAMTS-4. The expression of ADAMTS-4 was decreased significantly in the presence of NF-κB inhibitors. SIRT-1 activity was significantly downregulated in the macrophages and was reversed in the presence of the SIRT-1 agonist, resveratrol. Acetylation of NF-κB and hence the expression of ADAMTS-4 were significantly downregulated in the presence of SIRT-1 activator, resveratrol. Conclusions: Our study suggests that oxidized LDL significantly upregulated the expression of ADAMTS-4 in the monocytes/macrophages through ROS- NF-κB- SIRT-1 pathway.

Development of Selective ADAMTS-5 Peptide Substrates to Monitor Proteinase Activity.

The dysregulation of proteinase activity is a hallmark of osteoarthritis (OA), a disease characterized by progressive degradation of articular cartilage by catabolic proteinases such as a disintegrin and metalloproteinase with thrombospondin type I motifs-5 (ADAMTS-5). The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through in silico docking and combinatorial chemistry. The lead substrates 3 and 26 showed higher overall cleavage rates (∼3-4-fold) and catalytic efficiencies (∼1.5-2-fold) compared to the best current ADAMTS-5 substrate ortho-aminobenzoyl(Abz)-TESE↓SRGAIY-N-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH2. They exhibited high selectivity for ADAMTS-5 over ADAMTS-4 (∼13-16-fold), MMP-2 (∼8-10-fold), and MMP-9 (∼548-2561-fold) and detected low nanomolar concentrations of ADAMTS-5.

The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in ß3-ß4 (R756Q/R759Q/R762Q), ß9-ß10 (residues 828-835), and ß6-ß7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.

ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711.

Amyloid-ß (Aß) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aß(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aß1-42 ratio and Aß1-40/Aß1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aß levels. Finally, we found that the endogenous murine APP669-711/Aß1-42 ratio was increased in aged AD model mice, which shows Aß deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aß deposition in the brain of mouse models.

Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

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.

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.

Identification of Adamts4 as a novel adult cardiac injury biomarker with therapeutic implications in patients with cardiac injuries.

Pathological cardiac remodeling as an aftermath of a severe cardiac injury can lead to ventricular dysfunction and subsequent heart failure. Adamts4, a metalloproteinase, and disintegrin with thrombospondin-like motif, involved in the turnover of certain extracellular matrix molecules and pathogenesis of osteoarthritis, also plays a role in cardiac remodeling although little is presently known about its expression and function in the heart. Here, we have investigated the dynamic expression pattern of Adamts4 during cardiogenesis and also in the adult heart. To our surprise, adult cardiac injury reactivated Adamts4 expression concomitant with fibrosis induction. To better understand the mechanism, cultured H9c2 cardiomyocyte cells were subjected to ROS injury and Hypoxia. Moreover, through combinatorial treatment with SB431542 (an inhibitor of Tgf-ß1), and Adamts4 siRNA mediated gene knockdown, we were able to decipher a regulatory hierarchy to the signal cascade being at the heart of Tgf-ß regulation. Besides the hallmark expression of Adamts4 and Tgf-ß1, expression of other fibrosis-related markers like Collagen-III, alpha-SMA and Periostin were also assessed. Finally, increased levels of Adamts4 and alpha-SMA proteins in cardiac patients also resonated well with our animal and cell culture studies. Overall, in this study, we highlight, Adamts4 as a novel biomarker of adult cardiac injury.

The Role of ADAMTS-4 in Atherosclerosis and Vessel Wall Abnormalities.

Extracellular matrix proteins are regulated by metzincin proteases, like the disintegrin metalloproteinases with thrombospondin motifs (ADAMTS) family members. This review focuses on the emerging role which ADAMTS-4 might play in vascular pathology, which has implications for atherosclerosis and vessel wall abnormalities, as well as for the resulting diseases, such as cardiovascular and cerebrovascular disease, aortic aneurysms, and dissections. Major substrates of ADAMTS-4 are proteoglycans expressed physiologically in smooth muscle cells of blood vessels. Good examples are versican and aggrecan, principal vessel wall proteoglycans that are targeted by ADAMTS-4, driving blood vessel atrophy, which is why this metzincin protease was implicated in the pathophysiology of vascular diseases with an atherosclerotic background. Despite emerging evidence, it is important not to exaggerate the role of ADAMTS-4 as it is likely only a small piece of the complex atherosclerosis puzzle and one that could be functionally redundant due to its high structural similarity to other ADAMTS family members. The therapeutic potential of inhibiting ADAMTS-4 to halt the progression of vascular disease after initialization of treatment is unlikely. However, it is not excluded that it might find a purpose as a biomarker of vascular disease, possibly as an indicator in a larger cytokine panel.

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

The 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-1ß, tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), 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-ß 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-ß, or IL-1α, TNF-α and TGF-ß. The signaling molecules' inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-ß-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-ß-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-ß 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-ß 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.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support.

BACKGROUND: The effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood. OBJECTIVES: The authors sought to quantify indexes of fibrosis and determine the molecular signature of post-CF-LVAD vascular remodeling. METHODS: Paired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD-mediated changes in aortic mRNA and protein expression. RESULTS: The mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 µm pre-LVAD to 410 ± 209 µm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix-degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted. CONCLUSIONS: There is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix-degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.

Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.

Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that RORα, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of RORα is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of RORα, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD in vitro, tumor necrosis factor alpha (TNF-α) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF-α treatment. Next, TNF-α treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.

Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach.

The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SIGNIFICANCE: Versican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.

Conditioned medium of IGF1-induced synovial membrane mesenchymal stem cells increases chondrogenic and chondroprotective markers in chondrocyte inflammation.

Recently, mesenchymal stem cells (MSCs) have been the most explored cells for cell therapy for osteoarthritis (OA) that can be obtained from various sources. Synovial membrane MSCs (SMMSCs) provide best potential for OA therapy, however they are not widely explored. Conditioned medium of SMMSCs (SMMSCs-CM) rich in growth factors and cytokines can inhibit apoptosis and increase chondrocytes cell proliferation. The aim of the present study was to determine growth factors content in SMMSCs-CM as well as the chondrogenic and chondroprotective markers expression in OA model after insulin-like growth factor (IGF)1-induced and non-induced SMMSCs-CM treatments. Chondrocyte cell line (CHON002) was induced by IL1ß as OA model (CHON002 with IL1ß (IL1ß-CHON002)) and treated with SMMSCs-CM with or without IGF1 induction to determine its effectiveness in repairing OA cells model. ELISA was used to assay BMP2, fibroblast growth factor 18 (FGF18) and transforming growth factor (TGF) ß1 (TGFß1) levels in SMMSCs-CM, matrix metalloproteinase (MMP) 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) levels in OA cells model treated with SMMSCs-CM. RT-qPCR analyses were used to investigate the gene expression of SOX9, COL2, and COL10. CM from SMMSCs cultured and induced by IGF1 150 ng/mL was the most effective concentration for increasing the content of growth factor markers of SMMSCs-CM, which had successfully increased negative cartilage hypertrophy markers (SOX9 and COL2) and reduced hypertrophy markers (COL10, MMP13, and ADAMTS4). Preconditioning with IGF1 has better and very significant results in lowering MMP13 and ADAMTS4 levels. The present study supports IGF1 pre-conditioned SMMSCs-CM to develop a new therapeutic approach in OA improvement through its chondrogenic and chondroprotective roles.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Amelioration of Posttraumatic Osteoarthritis in Mice Using Intraarticular Silencing of Periostin via Nanoparticle-Based Small Interfering RNA.

OBJECTIVE: Recent evidence delineates an emerging role of periostin in osteoarthritis (OA), since its expression after knee injury is detrimental to the articular cartilage. We undertook this study to examine whether intraarticular (IA) knockdown of periostin would ameliorate posttraumatic OA in a murine model. METHODS: Posttraumatic OA was induced in 10-week-old male C57BL/6J mice (n = 24) by destabilization of the medial meniscus (DMM), and mice were analyzed 8 weeks after surgery. Periostin expression was inhibited by small interfering RNA (siRNA) delivered IA using a novel peptide-nucleotide polyplex. Following histologic assessment of the mouse knee cartilage, the extent of cartilage degeneration was determined using Osteoarthritis Research Society International (OARSI) cartilage damage score, and severity of synovitis was also assessed. Bone changes were measured using micro-computed tomography. The effect and mechanism of periostin silencing were investigated in human chondrocytes that had been stimulated with interleukin-1ß (IL-1ß) with or without the IκB kinase 2 inhibitor SC-514. RESULTS: Periostin expression in mice with posttraumatic OA was significantly abolished using IA delivery of a peptide-siRNA nanoplatform. OARSI cartilage damage scores were significantly lower in mice receiving periostin siRNA (mean ± SEM 10.94 ± 0.66) compared to untreated mice (22.38 ± 1.30) and mice treated with scrambled siRNA (22.69 ± 0.87) (each P = 0.002). No differences in the severity of synovitis were observed. Subchondral bone sclerosis, bone volume/total volume, volumetric bone mineral density, and heterotopic ossification were significantly lower in mice that had received periostin siRNA treatment. Immunostaining of cartilage revealed that periostin knockdown reduced the intensity of DMM-induced matrix metalloproteinase 13 (MMP-13) expression and also diminished the phosphorylation of p65 and immunoreactivity of the aggrecan neoepitope DIPEN. Periostin knockdown also suppressed IL-1ß-induced MMP-13 and ADAMTS-4 expression in chondrocytes. Mechanistically, periostin-induced MMP-13 expression was abrogated by SC-514, demonstrating a link between periostin and NF-κB. CONCLUSION: IA delivery of the periostin-siRNA nanocomplex represents a promising clinical approach to mitigate the severity of joint degeneration in OA. Our findings may thus provide an unequivocal scientific rationale for longitudinal studies of this approach. Utilizing a cartilage-specific gene-knockout strategy will further illuminate the functional role of periostin in OA.