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.

Amyloid-β (Aβ) 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. Aβ(-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/Aβ1-42 ratio and Aβ1-40/Aβ1-42 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 Aβ levels. Finally, we found that the endogenous murine APP669-711/Aβ1-42 ratio was increased in aged AD model mice, which shows Aβ 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 Aβ deposition in the brain of mouse models.

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.

Although NG2 is known to be selectively expressed in oligodendrocyte precursor cells (OPCs) for many years, its expressional regulation and functional involvement in oligodendrocyte differentiation have remained elusive. Here, we report that the surface-bound NG2 proteoglycan can physically bind to PDGF-AA and enhances PDGF receptor alpha (PDGFRα) activation of downstream signaling. During differentiation stage, NG2 protein is cleaved by A disintegrin and metalloproteinase with thrombospondin motifs type 4 (Adamts4), which is highly upregulated in differentiating OPCs but gradually downregulated in mature myelinating oligodendrocytes. Genetic ablation of Adamts4 gene impedes NG2 proteolysis, leading to elevated PDGFRα signaling but impaired oligodendrocyte differentiation and axonal myelination in both sexes of mice. Moreover, Adamts4 deficiency also lessens myelin repair in adult brain tissue following Lysophosphatidylcholine-induced demyelination. Thus, Adamts4 could be a potential therapeutic target for enhancing oligodendrocyte differentiation and axonal remyelination in demyelinating diseases.SIGNIFICANCE STATEMENT NG2 is selectively expressed in OPCs and downregulated during differentiation stage. To date, the molecular mechanism underlying the progressive removal of NG2 surface proteoglycan in differentiating OPCs has been unknown. In this study, we demonstrate that ADAMTS4 released by differentiating OPCs cleaves surface NG2 proteoglycan, attenuates PDGFRα signaling, and accelerates oligodendrocyte differentiation. In addition, our study also suggests ADAMTS4 as a potential therapeutic target for promoting myelin recovery in demyelinating diseases.

Aggrecan degradation is considered to play a key role in the progression of osteoarthritis (OA). Aggrecanases are members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and degrade aggrecan in OA cartilage. The aim of this study was to clarify the mechanisms of expression of ADAMTS4 induced by IL-1β in human fibroblast-like synoviocyte (HFLS) cells by high molecular weight hyaluronan (HMW-HA), a therapeutic agent used for OA. Monolayer cultures of HFLS cells were incubated with IL-1β and HMW-HA. In some experiments, cells were pretreated with the CD44 function-blocking monoclonal antibody or inhibitors of signaling pathways prior to addition of IL-1β and HMW-HA. The expressions of ADAMTS4 mRNA and protein were monitored using real-time RT-PCR, Western blotting, and immunofluorescence microscopy. To further determine the role of HMW-HA in IL-1β-induced ADAMTS4 expression, activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), Akt, and NF-κB were analyzed by Western blotting. HMW-HA suppressed ADAMTS4 mRNA and protein expressions induced by IL-1β. Pretreatment with the anti-CD44 monoclonal antibody recovered the inhibitory effect of HMW-HA on expression of ADAMTS4 mRNA induced by IL-1β. Western blotting analysis revealed that IL-1β-induced phosphorylation of p38 MAPK and JNK protein were diminished by HMW-HA. Furthermore, inhibition of the p38 MAPK and JNK pathways by chemical inhibitors suppressed ADAMTS4 mRNA expression stimulated by IL-1β. These results suggest that HMW-HA plays an important role as a regulatory factor in synovial tissue inflammation.

Chondroitin sulphate proteoglycans (CSPGs) are inhibitors to axon regeneration and plasticity. A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) is a human enzyme that catalyses the proteolysis of CSPG protein cores. Infusion of ADAMTS4 into the damaged spinal cord was previously shown to improve functional recovery SCI, however, this therapy is limited in its enzyme form. Adeno-associated viral (AAV) vector gene therapy has emerged as the vector of choice for safe, robust and long-term transgene expression in the central nervous system. Here, an AAV expression cassette containing ADAMTS4 under the control of the astrocytic GfaABC1D promoter was packaged into an AAV5 vector. Sustained expression of ADAMTS4 was achieved in vitro and in vivo leading to degradation of CSPGs. Compared to a contusion only group, AAV-ADAMTS4 resulted in significantly decreased lesion size, increased sprouting of hindlimb corticospinal tract axons, increased serotonergic fiber density caudal to a contusive spinal cord injury. Hindlimb-specific exercise rehabilitation was used to drive neuroplasticity towards improving functional connections. The combination of hindlimb rehabilitation with AAV-ADAMTS4 led to functional recovery after SCI compared to a contusion only group. Thus, long-term degradation of CSPGs through AAV-ADAMTS4 gene therapy in a combinational approach with rehabilitation represents a candidate for further preclinical development.

BACKGROUND Type A AAD, a serious cardiovascular emergency requiring urgent surgery, is the most common and serious AAD. The aim of this study was to investigate the diagnostic value of ADAMTS1 and ADAMTS4 in patients with type A acute aortic dissection (AAD). MATERIAL AND METHODS Immunohistochemistry and qRT-PCR were used to evaluate the protein and mRNA expression levels of ADAMTS1 and ADAMTS4 in 14 type A acute aortic dissection (AAD) tissues and 10 control aortic tissues. Serum ADAMTS1 and ADAMTS4 expression levels in 74 patients with type A AAD, 36 patients with hypertension (HPT), and 34 healthy donors were examined by ELISA. The diagnostic value of serum ADAMTS1 and ADAMTS4 were determined by receiver operator characteristic curve (ROC). Furthermore, the dynamic change of serum ADAMTS1, ADAMTS4, D-dimer, and CRP were detected before and after surgery at different time-points in 14 patients with type A AAD. RESULTS ADAMTS1 and ADAMTS4 protein and mRNA expression levels were found to be significantly higher in 14 type A AAD tissues (p<0.0001) compared with 10 control tissues. Serum ADAMTS1 and ADAMTS4 levels were significant higher in patients with type A AAD than those in the HPT and HD group (p<0.0001 for both). The AUC value, sensitivity, and specificity of ADAMTS1 were 0.9710 (95% CI: 0.9429 to 0.9991), 87.84%, and 97.06%, respectively, and those of ADAMTS4 were 0.9893 (95% CI: 0.9765 to 1.002), 94.59%, and 97.06%, respectively. In addition, serum ADAMTS4 level was gradually decreased with the time extension after surgery, similar to D-dimer change. CONCLUSIONS These data suggest that measurement of serum ADAMTS1 and ADAMTS4 levels could be potential diagnostic biomarkers for type A AAD, and ADAMTS4 might be a risk factor associated with type A AAD.

Evidence indicate that the brain-specific protein, brevican, is proteolytically cleaved during neurodegeneration, hence positioning fragments of brevican as potential blood biomarkers of neurodegenerative diseases, such as dementia. We aimed to develop two assays capable of detecting the brevican N-terminal (N-Brev) and the ADAMTS4-generated fragment (Brev-A), cleaved at Ser401, in serum and to perform a preliminary assessment of their diagnostic potential in dementias. Monoclonal antibodies against N-Brev and Brev-A were used to develop two ELISAs detecting each epitope. A comparison of brevican fragments in serum from individuals with AD (n = 28), other dementia (OD) (n = 41), and non-dementia-related memory complaints (NDCs) (n = 48) was conducted. Anti-N-Brev and anti-Brev-A antibodies selectively recognized their targets and dilution and spike recoveries were within limits of ±20%. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. For the N-Brev biomarker, serum from patients with OD showed significantly lower levels than those with AD (p = 0.05) and NDCs (p < 0.01). The opposite pattern was evident for Brev-A: serum levels in patients with OD were significantly higher than for AD (p = 0.04) and NDCs (p = 0.01). For both N-Brev and Brev-A, levels did not differ between AD and NDCs. The ratio of N-Brev/Brev-A resulted in increased significant differences between OD and AD (p < 0.01) and between OD and NDCs (p < 0.0001). The ratio discriminated between NDCs and OD (AUC: 0.75, 95% CI: 0.65-0.85, p < 0.0001) and between OD and AD (AUC: 0.72, 95% CI: 0.59-0.85, p < 0.01). In conclusion, we developed the first assays detecting the N-terminal of brevican as well as an ADAMTS4-cleaved fragment of brevican in blood. Differential levels of N-Brev and Brev-A between AD and OD allow for these biomarkers to possibly distinguish between different forms of dementias.

We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4_v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase.

Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the hypothesis that molecules whose expression is upregulated in injured pulmonary vascular endothelial cells (VECs) are involved in the pathogenesis of ARDS, we conducted a study to elucidate the molecular mechanisms and identify target factors for treatment. Primary human lung microvascular endothelial cells (HMVEC-Ls) were stimulated with lipopolysaccharide (LPS) or poly (I:C) and analyzed via a microarray to identify target genes for ARDS. We found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) was induced in murine lung VECs in an LPS-mediated ARDS model. Elevated ADAMTS4 was also observed by the immunostaining of lung samples from ARDS patients. The suppression of ADAMTS4 by siRNA in VECs ameliorated LPS-stimulated vascular permeability. The impairment of the cell surface expression of syndecan-1, a marker of the glycocalyx that is an extracellular matrix involved in vascular permeability, was dramatically inhibited by ADAMTS4 suppression. In addition, the suppression of ADAMTS4 protected against LPS-induced reductions in syndecan-1 and the adherens junction protein vascular endothelial cadherin. These results suggest that ADAMTS4 regulates VEC permeability in ARDS and may be a predictive marker and therapeutic target for ARDS.

Previous studies have shown that single-nucleotide polymorphisms (SNPs) of a disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (ADAMTS4) may involve in the pathogenesis of some diseases. However, it is not clear whether they are associated with hepatocellular carcinoma (HCC). A hospital-based case-control study, including 862 cases with HCC and 1120 controls, was conducted to assess the effects of 258 SNPs in the coding regions of ADAMTS4 on HCC risk and prognosis. We found that six SNPs in ADAMTS4 were differential distribution between cases and controls via the primary screening analyses; however, only rs538321148 and rs1014509103 polymorphisms were further identified to modify the risk of HCC (odds ratio: 2.73 and 2.95; 95% confidence interval, 2.28-3.29 and 2.43-3.58; P-value, 5.73 × 10-27 and 1.36 × 10-27 , respectively). Significant interaction between these two SNPs and two known causes of hepatitis B virus and aflatoxin B1 were also observed. Furthermore, rs538321148 and rs1014509103 polymorphisms were associated not only with clinicopathological features of tumor such as tumor stage and grade, microvessel density, and vessel metastasis, but with poor overall survival. Additionally, these SNPs significantly downregulated ADATMS4 expression in tumor tissues. These data suggest that SNPs in the coding region of ADAMTS4, such as rs538321148 and rs1014509103, may be potential biomarkers for predicting HCC risk and prognosis.

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine-inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine-stimulated chondrocytes and chondrocytic cells. Pre-incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine-stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra-articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size-dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247-3255, 2018.

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein-rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice-like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice-like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan-degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican-degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin-expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development.

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 β3-β4 (R756Q/R759Q/R762Q), β9-β10 (residues 828-835), and β6-β7 (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.

Seomae mugwort, a Korean native variety of Artemisia argyi, exhibits physiological effects against various diseases. However, its effects on osteoarthritis (OA) are unclear. In this study, a Seomae mugwort extract prevented cartilage destruction in an OA mouse model. In vitro and ex vivo analyses revealed that the extract suppressed MMP3, MMP13, ADAMTS4 and ADAMTS5 expression induced by IL-1β, IL-6 and TNF-α and inhibited the loss of extracellular sulphated proteoglycans. In vivo analysis revealed that oral administration of the extract suppressed DMM-induced cartilage destruction. We identified jaceosidin in Seomae mugwort and showed that this compound decreased MMP3, MMP13, ADAMTS4 and ADAMTS5 expression levels, similar to the action of the Seomae mugwort extract in cultured chondrocytes. Interestingly, jaceosidin and eupatilin combined had similar effects to Seomae mugwort in the DMM-induced OA model. Induction of IκB degradation by IL-1β was blocked by the extract and jaceosidin, whereas JNK phosphorylation was only suppressed by the extract. These results suggest that the Seomae mugwort extract and jaceosidin can attenuate cartilage destruction by suppressing MMPs, ADAMTS4/5 and the nuclear factor-κB signalling pathway by blocking IκB degradation. Thus, the findings support the potential application of Seomae mugwort, and particularly jaceosidin, as natural therapeutics for OA.

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Although Hif-2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif-2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif-2α-induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL-1β-, IL-6, IL-17- and TNF-α-induced up-regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX-2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif-2α, which directly up-regulates MMP3, MMP13, ADAMTS4, IL-6 and COX-2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif-2α expression and inhibited Hif-2α-induced MMP3, MMP13, ADAMTS4, IL-6 and COX-2 expression in articular chondrocytes. IL-1β induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif-2α expression, was completely blocked by apigenin in a concentration-dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif-2α inhibitors.

We investigated TNF-α and IL-1β regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-κB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-α and IL-1β treatment. Treatment of cells with MAPK and NF-κB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38α, p38β2, and p38γ were involved in induction, ERK2 and p38δ played no role in TNF-α-dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-α, and shIKK-β significantly decreased TNF-α-dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-α-dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-κB signaling, TNF-α and IL-1β modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro.

The actin cytoskeleton of trabecular meshwork (TM) cells is a therapeutic target for lowering intraocular pressure (IOP) in glaucoma patients. Netarsudil (the active ingredient in RhopressaTM) is a Rho-associated protein kinase inhibitor that induces disassembly of actin stress fibers. Here, we used live cell imaging of SiR-actin-labeled normal (NTM) and glaucomatous TM (GTM) cells to investigate actin dynamics during actin-driven biological processes with and without netarsudil treatment. Actin stress fibers were thicker in GTM than NTM cells and took longer (>120 min) to disassemble following addition of 1 µM netarsudil. Actin-rich extracellular vesicles (EVs) were derived by two mechanisms: exocytosis of intracellular-derived vesicles, and cleavage of filopodial tips, which detached the filopodia from the substratum, allowing them to retract to the cell body. While some phagocytosis was noted in untreated TM cells, netarsudil potently stimulated phagocytic uptake of EVs. Netarsudil treatment induced lateral fusion of tunneling nanotubes (TNTs) that connected adjacent TM cells; TNTs are important for TM cellular communication. Together, our results suggest that netarsudil may clear outflow channels in TM tissue by inducing phagocytosis and/or by modulating TM communication via EVs and TNTs. These cellular functions likely work together to regulate IOP in normal and glaucomatous TM.

Mechanical stimulation appears to play a key role in cartilage homeostasis maintenance, but it can also contribute to osteoarthritis (OA) pathogenesis. Accumulating evidence suggests that cartilage loading in the physiological range contributes to tissue integrity maintenance, whereas excessive or reduced loading have catabolic effects. However, how mechanical stimuli can regulate joint homeostasis is still not completely elucidated and few data are available on human cartilage. We aimed at investigating human OA cartilage response to ex vivo loading at physiological intensity. Cartilage explants from ten OA patients were subjected to ex vivo controlled compression, then recovered and used for gene and protein expression analysis of cartilage homeostasis markers. Compressed samples were compared to uncompressed ones in presence or without interleukin 1β (IL-1β) or interleukin 4 (IL-4). Cartilage explants compressed in combination with IL-4 treatment showed the best histological scores. Mechanical stimulation was able to significantly modify the expression of collagen type II (collagen 2), aggrecan, SOX9 transcription factor, cartilage oligomeric matrix protein (COMP), collagen degradation marker C2C and vascular endothelial growth factor (VEGF). Conversely, ADAMTS4 metallopeptidase, interleukin 4 receptor alpha (IL4Rα), chondroitin sulfate 846 epitope (CS846), procollagen type 2 C-propeptide (CPII) and glycosaminoglycans (GAG) appeared not modulated. Our data suggest that physiological compression of OA human cartilage modulates the inflammatory milieu by differently affecting the expression of components and homeostasis regulators of the cartilage extracellular matrix.