Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

Modulation of early osteoarthritis by tibiofemoral re-alignment in sheep.

OBJECTIVE: To investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA. METHOD: Normal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone. RESULTS: Multivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface-to-volume ratio (all P ≤ 0.0359 vs. normal). CONCLUSION: The data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

TMF inhibits extracellular matrix degradation by regulating the C/EBPß/ADAMTS5 signaling pathway in osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease, characterized by degenerative destruction of articular cartilage. Chondrocytes, the unique cell type in cartilage, mediate the metabolism of extracellular matrix (ECM), which is mainly constituted by aggrecan and type II collagen. A disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) is an aggrecanase responsible for the degradation of aggrecan in OA cartilage. CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor in the C/EBP family, has been reported to mediate the expression of ADAMTS5. Our previous study showed that 5,7,3',4'-tetramethoxyflavone (TMF) could activate the Sirt1/FOXO3a signaling in OA chondrocytes. However, whether TMF protected against ECM degradation by down-regulating C/EBPß expression was unknown. In this study, we found that aggrecan expression was down-regulated, and ADAMTS5 expression was up-regulated. Knockdown of C/EBPß could up-regulate aggrecan expression and down-regulate ADAMTS5 expression in IL-1ß-treated C28/I2 cells. TMF could compromise the effects of C/EBPß on OA chondrocytes by activating the Sirt1/FOXO3a signaling. Conclusively, TMF exhibited protective activity against ECM degradation by mediating the Sirt1/FOXO3a/C/EBPß pathway in OA chondrocytes.

REST, RCOR1 and RCOR2 expression is reduced in osteoarthritic chondrocytes and contributes to increasing MMP13 and ADAMTS5 expression through upregulating HES1.

Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells. Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII. These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.

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.

MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis.

Skeletal muscle, as a regenerative organization, plays a vital role in physiological characteristics and homeostasis. However, the regulation mechanism of skeletal muscle regeneration is not entirely clear. miRNAs, as one of the regulatory factors, exert profound effects on regulating skeletal muscle regeneration and myogenesis. This study aimed to discover the regulatory function of important miRNA miR-200c-5p in skeletal muscle regeneration. In our study, miR-200c-5p increased at the early stage and peaked at first day during mouse skeletal muscle regeneration, which was also highly expressed in skeletal muscle of mouse tissue profile. Further, overexpression of miR-200c-5p promoted migration and inhibited differentiation of C2C12 myoblast, whereas inhibition of miR-200c-5p had the opposite effect. Bioinformatic analysis predicted that Adamts5 has potential binding sites for miR-200c-5p at 3'UTR region. Dual-luciferase and RIP assays further proved that Adamts5 is a target gene of miR-200c-5p. The expression patterns of miR-200c-5p and Adamts5 were opposite during the skeletal muscle regeneration. Moreover, miR-200c-5p can rescue the effects of Adamts5 in the C2C12 myoblast. In conclusion, miR-200c-5p might play a considerable function during skeletal muscle regeneration and myogenesis. These findings will provide a promising gene for promoting muscle health and candidate therapeutic target for skeletal muscle repair.

Inhibition of SMAD3 effectively reduces ADAMTS-5 expression in the early stages of osteoarthritis.

OBJECTIVE: As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-ß signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown. METHODS: Sprague-Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3. RESULTS: In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete. CONCLUSION: The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.

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.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BACKGROUND: Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear. METHODS: Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis. RESULTS: The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-ß treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency. CONCLUSIONS: These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

lncRNA PSMB8-AS1 promotes colorectal cancer progression through sponging miR-1299 to upregulate ADAMTS5.

Long non-coding RNAs (lncRNAs) have been reported to be vital participants in tumor progression. Recently, lncRNA PSMB8-AS1 has been uncovered to facilitate pancreatic cancer progression by regulating miR-382-3p/STAT1/PD-L1 network. Nonetheless, the role of PSMB8-AS1 and its underlying mechanism have not been well-explored in colorectal cancer (CRC). The expression of RNAs or proteins was detected via qRT-PCR or western blot assays. Functional assays were involved in evaluating the effects of PSMB8-AS1/miR-1299/ADAMTS5 on the malignant behaviors of CRC cells. The molecular mechanism of PSMB8-AS1 was explored via mechanism analyses in CRC cells. Based on experimental results, PSMB8-AS1 expression was notably higher in CRC cell lines than in normal cells. The downregulation of PSMB8-AS1 repressed cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC while promoting cell apoptosis. It was also revealed that PSMB8-AS1 could sponge miR-1299 to upregulate ADAMTS5 in CRC cells. In rescue assays, we further discovered that miR-1299 inhibition or ADAMTS5 overexpression abrogated the suppressive influence of PSMB8-AS1 deficiency on CRC cell growth. In addition, PSMB8-AS1 was validated to induce M2 polarization. In conclusion, PSMB8-AS1 sponges miR-1299 to increase PSMB8-AS1 expression, thus promoting CRC cell growth.

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.

Silencing of circ_0000205 mitigates interleukin-1ß-induced apoptosis and extracellular matrix degradation in chondrocytes via targeting miR-766-3p/ADAMTS5 axis.

The aim of this study was to explore the role of hsa_circRNA_0000205 (circ_0000205) in chondrocyte injury in osteoarthritis (OA) and the underlying mechanism. Expression of circ_0000205, microRNA (miR)-766-3p and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 was detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot assays. Cell proliferation, apoptosis, and extracellular matrix (ECM) synthesis were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-ethynyl-2-deoxyuridine assays, flow cytometry, and qRT-PCR and Western blot assays. The target relationship between miR-766-3p and circ_0000205 or ADAMTS5 was confirmed by luciferase reporter assay and RNA immunoprecipitation. IL-1ß treatment could attenuate cell viability of primary chondrocytes and proliferating cell nuclear antigen (PCNA) and collagen II type alpha-1 (COL2A1) levels, and elevate apoptosis rate and cleaved caspase-3, ADAMTS5 and matrix metalloproteinase-13 (MMP13) levels, suggesting that IL-1ß induced chondrocyte apoptosis and ECM degradation. Expression of circ_0000205 was up-regulated in OA tissues and IL-1ß-induced primary chondrocytes, accompanied with miR-766-3p down-regulation and ADAMTS5 up-regulation. Knockdown of circ_0000205 could mitigate IL-1ß-induced above effects and improve cell proliferation. Moreover, both depleting miR-766-3p and promoting ADAMTS5 could partially counteract circ_0000205 knockdown roles in IL-1ß-cultured primary chondrocytes. Notably, circ_0000205 was verified as a sponge for miR-766-3p via targeting, and ADAMTS5 was a direct target for miR-766-3p. Silencing circ_0000205 could protect chondrocytes from IL-1ß-induced proliferation reduction, apoptosis, and ECM degradation by targeting miR-766-3p/ADAMTS5 axis.

Knockdown of circ-PRKCH alleviates IL-1ß-treated chondrocyte cell phenotypic changes through modulating miR-502-5p/ADAMTS5 axis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation. Circular RNAs (circRNAs) are involved in the initiation and development of OA. This study aimed to explore the potential role and mechanism of circRNA protein kinase C eta (circ-PRKCH) in OA. METHODS: A total of 30 cartilage specimens were collected from OA patients or normal subjects. Human chondrocytes (CHON-001) were stimulated with interleukin-1ß (IL-1ß) to establish an in vitro OA model. The expression levels of circ-PRKCH, microRNA-502-5p (miR-502-5p) and circ-PRKCH or A disintegrin and metalloproteases metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) in cartilage specimens and IL-1ß-treated chondrocytes were detected by quantitative real-time PCR or Western blot, and their correlation in OA cartilage specimens was analysed by Spearman's correlation coefficient. The targeted relationship between miR-502-5p and circ-PRKCH or ADAMTS5 was verified by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EDU), flow cytometry, wound healing and enzyme-linked immunosorbent assay (ELISA) assays were applied to evaluate cell proliferation, apoptosis, migration and inflammatory response in IL-1ß-treated chondrocytes. Exosomes were identified by transmission electron microscope (TEM) and Western blot. RESULTS: Circ-PRKCH and ADAMTS5 expression levels were up-regulated, while miR-502-5p expression was down-regulated in OA cartilage tissues and IL-1ß-treated chondrocytes. Depletion of circ-PRKCH relieved IL-1ß-treated chondrocyte cell phenotypic changes by promoting cell proliferation and migration, as well as inhibiting apoptosis and inflammatory response. Mechanically, circ-PRKCH acted as a sponge for miR-502-5p to regulate ADAMTS5 expression, thereby contributing to IL-1ß-treated chondrocyte cell phenotypic changes. Moreover, exosomes derived from IL-1ß-treated chondrocytes could transfer circ-PRKCH across cells. CONCLUSION: Circ-PRKCH contributed to IL-1ß-treated cell phenotypic changes in chondrocytes via modulating miR-502-5p/ADAMTS5 pathway, which might provide a promising biomarker for OA treatment.

MicroRNA-148a targets ADAMTS5 to inhibit proliferation of endometriosis cells.

To examine miR-148a expression in the serum of patients with endometriosis (EMS), and to further explore the target of miR-148a in HS832.Tc cells and the effect of miR-148a on the proliferation of Hs832.Tc cells. The serum of non-EMS patients and EMS patients were collected and real-time quantitative PCR (qRT-PCR) was used to detect miR-148a in serum. The EMS cell line Hs832.Tc was cultured and transfected with miR-148aminic, miR-148a inhibitor to construct over expressing and interfering cell lines. Cell viability and apoptosis were detected. The dual luciferase assay identified a target relationship between miR-148a and ADAMTS5 and whether miR-148a regulates proliferation of endometriosis cells via ADAMTS5 was further validated. Compared with normal subjects, miR-148a was significantly reduced in serum of EMS patients (p<0.05). The area under the receiver operating characteristic curve for miR-148a for diagnosis of EMS was 0.91, which was statistically significant (p<0.01). The proliferation of Hs832.Tc cells was significantly inhibited and the cell apoptosis was increased after miR-148a over expression. The proliferation of Hs832.Tc cells was promoted and apoptosis was reduced by miR-148a down regulation. The dual luciferase report demonstrates that ADAMTS5 is a target gene of miR-148a. The addition of ADAMTS5 can directly promote apoptosis of Hs832.Tc cells. The expression of miR-148a in serum of EMS patients was decreased, and miR-148a targets ADAMTS5 to promote apoptosis in EMS cells.

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

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

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

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

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

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

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Fibroblast growth factor 21 (FGF21) alleviates senescence, apoptosis, and extracellular matrix degradation in osteoarthritis via the SIRT1-mTOR signaling pathway.

Osteoarthritis (OA) is a complex condition that involves both apoptosis and senescence and currently cannot be cured. Fibroblast growth factor 21 (FGF21), known for its role as a potent regulator of glucose and energy metabolism, protects from various diseases, possibly by mediating autophagy. In the present study, the role of FGF21 in the progression of OA was investigated in both in vitro and in vivo experiments. In vitro, the results revealed that FGF21 administration alleviated apoptosis, senescence, and extracellular matrix (ECM) catabolism of the chondrocytes induced by tert-butyl hydroperoxide (TBHP) by mediating autophagy flux. Furthermore, CQ, an autophagy flux inhibitor, could reverse the protective effect of FGF21. It was observed that the FGF21-induced autophagy flux enhancement was mediated by the nuclear translocation of TFEB, which occurs due to the activation of the SIRT1-mTOR signaling pathway. The in vivo experiments demonstrated that FGF21 treatment could reduce OA in the DMM model. Taken together, these findings suggest that FGF21 protects chondrocytes from apoptosis, senescence, and ECM catabolism via autophagy flux upregulation and also reduces OA development in vivo, demonstrating its potential as a therapeutic agent in OA.

SNHG5 protects chondrocytes in interleukin-1ß-stimulated osteoarthritis via regulating miR-181a-5p/TGFBR3 axis.

Long noncoding RNAs (lncRNAs) have been considered as important modulators in the development of osteoarthritis. The present study investigates whether there is a link between lncRNA small nucleolar RNA host gene 5 (SNHG5) and osteoarthritis pathogenesis, and the underlying molecular mechanism. To establish an in vitro model of osteoarthritis, interleukin 1ß (IL-1ß) was used to treat chondrocytes (C20/A4 cells) for mimicking the inflammatory condition in osteoarthritis pathogenesis. SNHG5 and miR-181a-5p expression levels were then detected in cartilage tissues of osteoarthritis patients and C20/A4 cells by quantitative polymerase chain reaction (qPCR). Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were applied for detecting the viability of chondrocytes, and the apoptosis of chondrocytes was examined through caspase-3 activity assay and flow cytometry analysis. Western blot and qPCR were employed for determining the expression levels of TGFBR3, ADAMTS5, and MMP-13. The regulatory relationships among SNHG5, miR-181a-5p, and TGFBR3 were verified by RNA immunoprecipitation and dual-luciferase reporter assays. The expression levels of SNHG5 and TGFBR3 were markedly decreased, and miR-181a-5p expression was enhanced in osteoarthritis tissues and chondrocytes treated with IL-1ß. SNHG5 knockdown inhibited the viability of chondrocytes, induced apoptosis, and promoted the expression levels of ADAMTS5 and MMP-13. Conversely, SNHG5 overexpression could counteract the effects of IL-1ß, increase the viability of chondrocytes and suppress apoptosis. Mechanically, SNHG5 positively regulated TGFBR3 expression via sponging miR-181a-5p. Moreover, miR-181a-5p overexpression and TGFBR3 knockdown counteracted the effects of SNHG5 on chondrocytes. SNHG5 can probably protect chondrocytes from the inflammatory response and reduce the degradation of the extracellular matrix via modulating the miR-181a-5p/TGFBR3 axis.