ADAMTS4 Levels Research Articles

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Effects of occlusion modification on the remodelling of degenerative mandibular condylar processes.

The temporomandibular joint (TMJ) displays a high remodelling capability in response to occlusion changes. The purpose of the current study was to investigate the responses of TMJ condyles of growing mice to the installation of a unilateral anterior crossbite (UAC) prosthesis and the replacement of the UAC prothesis with a bilateral anterior elevation (BAE) prosthesis. C57BL/6J mice were randomly assigned to the blank control and experimental groups. In mice in the experimental groups, UAC was created, while in others, BAE was created after the creation of UAC or removal of UAC. Changes in TMJ condylar cartilage and subchondral bone were assessed. The degradation of condylar cartilage induced by UAC was reversed by BAE, as evaluated by cartilage histochemical changes, collagen II-positive area, collagen X-positive chondrocytes and expression levels of Adamts-5, Mmp13, Tnf-α and Il-1β. Subchondral bone was assessed based on the subchondral bone volume, the number of TRAP-positive cells and the Opg/Rankl ratio. The growing mouse TMJ condyle displays a high remodelling capability, which can be degenerative or rehabilitative in response to the creation of UAC and the replacement of UAC with BAE. Early correction of occlusion is beneficial for the recovery of degenerative condyles.

ADAMTS1 and HSPG2 mRNA levels in cumulus cells are related to human oocyte quality and controlled ovarian hyperstimulation outcomes

PurposeThe study investigated potential correlations between the expression levels of ADAMTS1 and HSPG2 in cumulus cells (CCs) and controlled ovarian hyperstimulation (COH) outcomes.MethodsRT-PCR was used to determine ADAMTS1 and HSPG2 mRNA levels in mice CCs at different timepoints (0, 4, 8, 12, and 16 h) after human chorionic gonadotropin (hCG) injection, and in CCs after RNAi treatment. Women with polycystic ovary syndrome (PCOS) (n = 45) and normal ovulatory controls (n = 103) undergoing IVF/ICSI were recruited. Relative ADAMTS1 and HSPG2 mRNA levels were measured by RT-PCR. Moreover, correlations of ADAMTS1 and HSPG2 levels with COH outcomes were analyzed.ResultsAt different timepoints after hCG treatment, ADAMTS1 mRNA had the highest level at 12 h, whereas HSPG2 showed opposite profiles to ADAMTS1 with the lowest level at 12 h. HSPG2 expression was upregulated after ADAMTS1 RNAi treatment The PCOS group had higher HSPG2 and lower ADAMTS1 expression levels than controls. In normal ovulatory women (control group), a higher expression of ADAMTS1 and lower expression of HSPG2 were associated with more mature oocytes, transplantable embryos, and good quality embryos, whereas higher transplantable embryo rates and good quality embryo rates were obtained only with lower HSPG2 expression. ROC curves showed the co-measurement of ADAMTS1 and HSPG2 had a better predictive power than separate analyses.ConclusionThe dynamic profiles of ADAMTS1 and HSPG2 were inversely correlated in CCs. In PCOS and normal ovulatory patients, higher ADAMTS1 and lower HSPG2 expression levels in CCs were related to better COH outcomes.

NEAT1/miR-193a-3p/SOX5 axis regulates cartilage matrix degradation in human osteoarthritis.

Long non-coding RNAs (lncRNAs) were reported to be involved in the progression of osteoarthritis (OA). The aim of this work was to explore the functional role of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in OA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was employed to analyze the expression of microRNA (miR-193a)-3p, NEAT1, and sex-determining region Y-box protein 5 (SOX5), as well as the levels of pro-inflammatory cytokines interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), and IL-8 in OA cartilage tissue and chondrocytes. In addition, flow cytometry was used to measure the apoptosis of chondrocytes. The protein levels of extracellular matrix ACAN, collagen type II α1 chain (Col2a1), matrix metalloproteinase-3 (MMP-3), MMP-13, a disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and SOX5 were determined using western blot analysis. Dual-luciferase reporter assay was performed to determine the target relationship among NEAT1, miR-193a-3p, and SOX5. We found that miR-193a-3p expression was downregulated, while NEAT1 and SOX5 were upregulated in OA cartilage tissue and chondrocytes. Both upregulation of miR-193a-3p and knockdown of NEAT1 suppressed inflammation, apoptosis, and reduced the protein levels of MMP-3, MMP-13, and ADAMTS-5, while elevating ACAN and Col2a1 expression in chondrocytes. NEAT1 targeted miR-193a-3p, and SOX5 was targeted by miR-193a-3p. Silencing of miR-193a-3p reversed the NEAT1 knockdown-mediated effect on the inflammation, apoptosis, and production of the extracellular matrix. The introduction of SOX5 abolished the impact of the upregulation of miR-193a-3p on inflammation, apoptosis, and production of extracellular matrix in chondrocytes. In conclusion, NEAT1/miR-193a-3p/SOX5 axis regulates cartilage matrix degradation in human OA.

ADAMTS-5 Decreases in Coronary Arteries and Plasma from Patients with Coronary Artery Disease.

The current study demonstrates that a disintegrin and metalloproteinase with thrombospondin type 1 motif- (ADAMTS-) 5 is a key extracellular matrix protease and associated with cardiovascular diseases. However, the plasma ADAMTS-5 levels and relevance of coronary artery disease (CAD) remain largely unknown. This study is aimed at examining the relationship between the plasma ADAMTS-5 levels and the severity of coronary stenosis in patients with CAD. In the present study, the expression of ADAMTS-5 was analyzed in coronary artery samples and blood. The results showed that the plasma ADAMTS-5 levels were lower in the CAD group than in the control group. In addition, significantly higher matrix metalloproteinase- (MMP-) 2 and MMP-9 levels were also observed in the patients with CAD, and the ADAMTS-5 levels were negatively correlated with the MMP-2 and MMP-9 levels. Spearman's correlation analysis showed that the Gensini score was negatively correlated with the ADAMTS-5 levels but was positively correlated with the MMP-2 and MMP-9 levels. Receiver-operating characteristic (ROC) analysis revealed that ADAMTS-5, MMP-2, and MMP-9 may have a certain diagnostic value in CAD and that the combination of all three metalloproteinases had a higher diagnostic value. The findings provided a better understanding of the role of ADAMTS-5 in the diagnosis of CAD.

Substantive molecular and histological changes within the meniscus with tears

BackgroundThe meniscus plays a vital role in the normal biomechanics of the knee. However, it is not well studied at the molecular level. The purpose of this study was to determine whether molecular and pathological changes in the meniscal tissue vary depending on the presence or absence of meniscal and/or anterior cruciate ligament tear (ACL).MethodsSix normal menisci (group A), seven simple torn menisci (group B) and seven torn menisci with concomitant anterior cruciate ligament tears (group C) were collected. We observed the pathological changes in the menisci and used real-time polymerase chain reaction along with immunohistochemistry and in situ hybridisation to examine the levels of ACAN, ADAMTS5, COL10A1, CEBPβ, MMP13 and miR-381-3p, miR-455-3p, miR-193b-3p, miR-92a-3p, respectively. Patients were scored preoperatively and postoperatively using the Lysholm Knee Scoring Scale and International Knee Documentation Committee Subjective Knee Evaluation Form.ResultsCompared with group A, the expression levels of ADAMTS5, COL10A1, CEBPβ, and MMP13 and all the miRNAs were increased while ACAN was down-regulated in groups B and C. Additionally, the gene expression and miRNA levels were higher in group C than that in group B, except for ACAN, which was lower. Several fibrochondrocytes strongly expressed ADAMTS5, CEBPβ, and MMP13 in groups B and C and had high levels of miR-381-3p and miR-455-3p than that in group A. Postoperative Lysholm and IKDC scores were higher in group B than in group C.ConclusionsOur findings suggest that the meniscus tended to degenerate after it was injured, especially when combined with a torn ACL. The miRNAs investigated in this study might also contribute to meniscus degeneration. Patients with a combined injury patterns might have relatively worse joint function.

MicroRNA-149 Suppresses Inflammation in Nucleus Pulposus Cells of Intervertebral Discs by Regulating MyD88.

BackgroundIntervertebral disc degeneration (IDD) is associated with low back and neck pain, but the mechanisms underlying its pathogenesis are unclear. In this study, we explored the function of microRNA-149 (miR-149) in inflammatory response mediated by lipopolysaccharide (LPS) in nucleus pulposus (NP) cells.Material/MethodsQuantitative real-time PCR was used to detect miRNA and mRNA levels, while Western blotting was utilized to determine protein levels. ELISA was used to examine chemokine production. The correlation between miR-149 and MyD88 was assessed by reporter assay. Apoptosis was examined by flow cytometry.ResultsmiR-149 expression was significantly decreased after LPS exposure in NP cells. Overexpression of miR-149 reversed LPS-induced inhibition in aggrecan and collagen II expression and attenuated LPS-mediated promotion in the levels of MMP3, ADAMTS4, and inflammatory cytokines. Moreover, we found that miR-149 exerted its function by targeting MyD88 in NP cells.ConclusionsmiR-149 can inhibit the inflammatory response mediated by LPS in NP cells, and might be a potential target for the treatment of IDD.

Desumoylation of aggrecan and collagen II facilitates degradation via aggrecanases in IL-1β-mediated osteoarthritis.

Background: Aggrecan plays a crucial role in the ability of tissues to withstand compressive loads during the pathological progression of osteoarthritis (OA). Progressive loss of aggrecan from cartilage may result in exposure of the collagen matrix and can lead to its disintegration by metalloproteases. Although aggrecanases are expressed constitutively in human chondrocytes, the degradation of aggrecan is induced by proinflammatory cytokines; however, little is known about the underlying mechanisms.Methods: Human primary chondrocytes from OA patients or healthy donors and human chondrogenic SW1353 cells were cultured and stimulated with IL-1β in vitro, the mRNA expressions and protein levels of MMP-13, ADAMTS-4, ADAMTS-5, SENP1, and SENP2 were determined using real time PCR and Western blot, respectively. The localizations of aggrecan and Col-II, as well as the SUMOylation modification of these proteins were analyzed using immunofluorescence and immunoprecipitation assays, respectively.Results: Our results showed that a proinflammatory cytokine interleukin-1β induced the OA model and desumoylation of aggrecan and collagen type II because the small ubiquitin-like modifier 2/3 (SUMO2/3) was co-localized with aggrecan and collagen type II proteins and interacted physically with them. Mechanistic studies have shown that knockdown of SUMO2/3 expression can significantly enhance the rate of degradation of aggrecan and collagen type II at both the mRNA and protein levels in the OA model. In addition, SUMO-specific protease 2 (SENP2) plays important roles in the desumoylation of aggrecan, while knockdown of SENP2 can protect aggrecan and collagen type II. Clinical assays have shown that OA patients have higher SENP2 levels than healthy controls, and the SENP2 level correlates negatively with both aggrecan and collagen type II levels.Conclusion: SENP2 desumoylates aggrecan and collagen type II proteins in the inflammation induced OA, and SENP2 expression correlates with OA progression.

LncRNA MALAT1 promotes osteoarthritis by modulating miR-150-5p/AKT3 axis

BackgroundMany studies have reported that long noncoding RNAs (lncRNAs) could act as sponges for microRNAs (miRNAs) and play important roles in the regulation of osteoarthritis (OA). Yet, the underlying mechanisms of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OA are still unclear. Therefore, we aimed to explore the regulation mechanisms of MALAT1 in OA procession.MethodsIL-1β treatment in chondrocyte was used to mimic OA in vitro. MALAT1, miR-150-5p and AKT3 expression levels were detected via qRT-PCR. The protein levels of AKT3, MMP-13, ADAMTS-5, Bax, Bcl-2, cleaved-PARP, collagen II and aggracan were measured by western blot. MTT assay was performed to detect cell proliferation ability. The apoptosis of chondrocytes was determined using flow cytometry and western blot. Luciferase assay and RNA immunoprecipitation (RIP) assays were used to confirm the relationship among MALAT1, miR-150-5p and AKT3.ResultsIn our study, MALAT1 and AKT3 were upregulated while miR-150-5p was downregulated in OA in vitro and vivo. The level of miR-150-5p was negatively correlated with that of MALAT1 or AKT3. More importantly, overexpression of MALAT1 promoted the expression of AKT3 by negatively regulating miR-150-5p. MALAT1 knockdown inhibited cell proliferation, promoted apoptosis, increased MMP-13, ADAMTS-5 expression and decreased collagen II, aggracan expression in IL-1β treated chondrocytes. MALAT1 upregulation or AKT3 overexpression enhanced proliferation, inhibited apoptosis and extracellular matrix (ECM) degradation, which was undermined by overexpression of miR-150-5p. By contrast, miR-150-5p depletion rescued the effect of MALAT1 downregulation or loss of AKT3 on IL-1β-stimulated chondrocytes.ConclusionMALAT1 was responsible for cell proliferation, apoptosis, and ECM degradation via miR-150-5p/AKT3 axis.

Elevated expression of hypoxia-inducible factor-2alpha regulated catabolic factors during intervertebral disc degeneration

Headings aimsThe present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. Materials and methodsHuman degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. Key findingsWe found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SignificanceHIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.

A disintegrin‐like and metalloproteinase with thrombospondin‐1 (ADAMTS‐1) levels in gingival crevicular fluid correlate with vascular endothelial growth factor‐A, hypoxia‐inducible factor‐1α, and clinical parameters in patients with advanced periodontitis

ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin) are a family of proteinases that are structurally similar to the family of matrix metalloproteinases with critical roles in damage and repair of the extracellular matrix. Their functions are closely related to inflammation, hypoxia, and vascularization. Our aim was to determine levels of ADAMTS-1 in gingival crevicular fluid (GCF) in patients with advanced periodontal diseases and identify their association with hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF-A), and clinical parameters of periodontitis. The study consisted of three groups: healthy individuals (control; n=20), generalized chronic periodontitis (CP; n=21), and generalized aggressive periodontitis (GAgP; n=20). Clinical parameters were measured. Levels of ADAMTS-1, VEGF-A, and HIF-1α in GCF and serum were quantified by enzyme-linked immunosorbent assay (ELISA) and reported as total amounts and concentration. ADAMTS-1 total amount in GCF were significantly higher in patients with CP and GAgP compared with healthy individuals (P<0.05). HIF-1α total amount in GCF were also higher in periodontitis groups compared with the control group (P<0.05). GCF total VEGF-A content was significantly higher in the GAgP group compared with the CP and the controls (respectively; P=0.023, P=0.003). There was a significant correlation between ADAMTS-1, VEGF-A, and HIF-1α levels in the GCF and clinical periodontal parameters (probing depth [PD], bleeding on probing [BOP], and clinical attachment loss (CAL); P<0.05). ADAMTS-1 may play a role in advanced periodontal disease pathogenesis in correlation with tissue hypoxia and vascularization.

Trans-Cinnamaldehyde Inhibits IL-1β-Stimulated Inflammation in Chondrocytes by Suppressing NF-κB and p38-JNK Pathways and Exerts Chondrocyte Protective Effects in a Rat Model of Osteoarthritis

Objective Trans-cinnamaldehyde (TCA), a compound from Cinnamomum cassia Presl, has been reported to have anti-inflammatory effect. However, its effect on cartilage degradation in osteoarthritis is unclear. This study is designed to examine the effects of TCA on cartilage in vitro and in vivo. Material and Methods SW1353 cells and human primary chondrocytes were treated with varying concentrations of TCA (2-20 μg/ml) for 2 h followed by IL-1β stimulation. Cell viability was examined by the MTT assay. Expression of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 was examined by Western blot and RT-qPCR. Monosodium iodoacetate (MIA)-induced OA was established in rats to assess the chondrocyte protective effects of intraperitoneal injection of TCA (50 mg/kg). Results TCA at a concentration of 10 μg/ml had no significant effect on cell viability. MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 were decreased by TCA 2-10 μg/ml in a dose-dependent manner (all P<0.05). Pretreatment with TCA decreased the degradation of IκBα and increased the expression of p-IκBα, indicating that NF-κB inactivation was induced by TCA in IL-1β-stimulated SW1353 cells. Pretreatment with TCA decreased the levels of p-p38 and p-JNK, while the levels of p-ERK were not significantly affected. TCA 10 μg/ml significantly decreased expression levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5. In vivo results showed that TCA alleviated cartilage destruction and the OARSI scores. Conclusion TCA possesses anti-inflammatory effect in vitro and exerts chondrocyte protective effects in vivo, in which NF-κB and p38-JNK were involved.

The Effect of Mesenchymal Stem Cell Wharton's Jelly on ADAMTS-4 and iNOS Levels in Osteoarthritis Rat Model.

BACKGROUND:Osteoarthritis (OA) is one of the most common diseases among the elderly. OA occurs due to an imbalance between degradation and synthesis in articular joint tissue, causing changes in joint components such as cells, matrices and molecular production. Therefore, knowledge of cartilage-degrading enzymes such as ADAMTS-4 and iNOS is needed.AIM:This study aims to prove the effect of Mesenchymal Stem Cell Wharton Jelly on decreasing ADAMTS-4 levels as cartilage-degrading enzymes and increasing levels of iNOS which showed the immunosuppressive potential of MSC-WJ in cases of osteoarthritis in vivo.MATERIAL AND METHODS:This research is an experimental study with the design of Post-test-Only Control Group Design. The sample consisted of 16 OA rats as a control group and 16 OA rats treated with MSC-WJ as a treatment group. OA induction is done by injection of monosodium iodoacetate (MIA) into the intra-articular right knee. Giving MSC-WJ is done in the third week after MIA induction. The serum ADAMTS-4 and iNOS levels were measured after 3 weeks treated with MSC-WJ using the ELISA method. The statistical test used is an independent t-test. The value of p < 0.05 was said to be statistically significant.RESULT:The result showed that serum ADAMTS-4 levels were lower in the group treated with MSC-WJ than in the control group, but not statistically significant (p > 0.05). Serum iNOS levels were higher in the group treated with MSC-WJ than in the control group (p < 0.05).CONCLUSION:This study concluded that MSC-WJ significantly reduced ADAMTS-4 levels and increased the serum iNOS levels of OA rats.

Role of stem cells and bioactive scaffold in chronic joint injury: Working towards a regenerative medicine approach to stop osteoarthritis progression

Purpose: Osteoarthritis is a leading cause of disability in aging individuals. Although commonly considered an age-related condition, a major predisposing factor is sports or trauma related joint injury sustained during young adulthood. Current treatments for joint injuries may help to relieve symptoms, but have little effect in preventing the development of chronic joint disease and progression to osteoarthritis later in life. Despite this being a significant clinical problem, the mechanism of impaired healing following joint injury, even when there is surgical intervention, remains poorly understood. This study tests the hypotheses that (1) impaired healing following joint injury is due to the inflammatory and highly inhibitory environment created by diseased cells, (2) this inhibitory environment can be reversed by ‘priming’ the diseased cells using human mesenchymal stem cells (hMSCs), which have documented anti-inflammatory and trophic properties, and (3) a bioactive scaffold can further promote regeneration in the injured joint. This study will aid the development of a novel regenerative therapy for joint injury, by restoring a joint environment that is conducive to healing and reducing the long-term risk of developing osteoarthritis. Methods: An in vitro model mimicking chronic joint injury was developed by co-culturing bone marrow-derived hMSCs with human synovial fibroblasts (HSFs) isolated from osteoarthritic joint tissues. To test hypothesis (1), the effects of HSFs on hMSCs were examined for 3, 7, 10 and 21 days in growth, osteogenic and chondrogenic media, which simulated the types of conditions present in the post-injury joint environment. To test hypothesis (2), the effects of hMSCs on HSFs were examined for 3 and 7 days in growth medium. To test hypothesis (3), the same set-up was used as for (1), with or without the addition of a bioactive scaffold composed of ‘Sr-HT-Gahnite’ ceramic (AU patent #2012262674; co-invented by J. Li). Quantitative RT-PCR was used to evaluate inflammatory response in the hMSCs and HSFs (IL-6, IL-8, MMP-1, MMP-2, MMP-13, ADAMTS4, ADAMTS5, CD44, TLR4, COX-2, CCL2), as well as differentiation in the hMSCs (RUNX2, BSP, SPP1, BGLAP for osteogenesis; SOX9, COL2A1, ACAN, TGFb1 for chondrogenesis), at all time points. Histology and histochemistry were performed at 21 days to compare osteogenesis and chondrogenesis in hMSCs between groups. Data was collected from 4 independent samples in each treatment group, and analysed using t-tests with p < 0.05 considered statistically significant. Results: The hMSCs showed significant upregulation of several inflammatory markers when co-cultured with diseased HSFs over a 21 day period. Notably, MMP-13 and TLR4 were upregulated at 7 days in all three media types, while IL-8, MMP-2, ADAMTS-5 and CD44 showed long-term upregulation at 21 days in at least one media type. Osteogenic and chondrogenic differentiation of hMSCs to form new tissues were both significantly impaired in the presence of diseased HSFs, as seen through reduced BSP and SPP1 expression and Alizarin red S staining for calcium (osteogenic markers), as well as reduced COL2A1 and ACAN expression and toluidine blue staining for proteoglycans (chondrogenic markers) at 21 days (Fig 1). When diseased HSFs were co-cultured with hMSCs, there was rapid downregulation of several inflammatory markers, including significant reduction in the levels of ADAMTS5, MMP-13 and TLR4, all of which are implicated in inflammation, matrix degradation and tissue degeneration. When a bioactive scaffold was added into the co-culture system, it showed simultaneous modulation of the inflammatory response in hMSCs and diseased HSFs through significant downregulation of ADAMTS-4, MMP-13 and CCL-2. Furthermore, the scaffold partly ‘rescued’ the impaired differentiation seen in hMSCs when co-cultured with diseased HSFs at 21 days, where the presence of the scaffold improved calcium (for osteogenesis) and proteoglycan (for chondrogenesis) deposition in the extracellular matrix (Fig 1). Conclusions: The findings suggest that diseased cells arising from joint injury can create a highly inhibitory environment that increases inflammation in stem cells normally involved in the repair of musculoskeletal tissues, which impairs their regenerative ability. This inhibitory environment can be reversed by ‘priming’ the injured joint prior to treatment, such as through an intra-articular injection of hMSCs, which can help modulate the pro-inflammatory state of the resident joint tissues. A Sr-HT-Gahnite bioactive scaffold may complement the effects of hMSCs to further promote regeneration in the joint. This novel regenerative medicine approach of combining stem cells and a bioactive scaffold may be useful as a new therapy to treat chronic joint injury and reduce the risk of osteoarthritis progression.

PNNS-Conjugated Chitosan Mediated IGF-1 and miR-140 Overexpression in Articular Chondrocytes Improves Cartilage Repair

The aim of the present study was to investigate the effects of phosphorylatable nucleus localization signal linked nucleic kinase substrate short peptide (pNNS)-conjugated chitosan (pNNS-CS) mediated miR-140 and IGF-1 in both rabbit chondrocytes and cartilage defects model. pNNS-CS was combined with pBudCE4.1-IGF-1, pBudCE4.1-miR-140, and negative control pBudCE4.1 to form pDNA/pNNS-CS complexes. Then these complexes were transfected into chondrocytes or injected intra-articularly into the knee joints. High levels of IGF-1 and miR-140 expression were detected both in vitro and in vivo. Compared with pBudCE4.1 group, in vitro, the transgenic groups significantly promoted chondrocyte proliferation, increased glycosaminoglycan (GAG) synthesis, and ACAN, COL2A1, and TIMP-1 levels, and reduced the levels of nitric oxide (NO), MMP-13, and ADAMTS-5. In vivo, the exogenous genes enhanced COL2A1, ACAN, and TIMP-1 expression in cartilage and reduced cartilage Mankin score and the contents of NO, IL-1β, TNF-α, and GAG contents in synovial fluid of rabbits, MMP-13, ADAMTS-5, COL1A2, and COL10A1 levels in cartilage. Double gene combination showed better results than single gene. This study indicate that pNNS-CS is a better gene delivery vehicle in gene therapy for cartilage defects and that miR-140 combination IGF-1 transfection has better biologic effects on cartilage defects.

Associations between second-trimester amniotic fluid levels of ADAMTS4, ADAMTS5, IL-6, and TNF-α and spontaneous preterm delivery in singleton pregnancies.

Background We investigated the roles of inflammatory cytokines and the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family in the etiopathogenesis of spontaneous preterm delivery by comparing the ADAMTS4, ADAMTS5, interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) levels in second-trimester amniotic fluid between pregnant women with preterm birth and term controls. Methods All pregnant women who underwent second-trimester amniocentesis for genetic analysis between January 1, 2016, and January 1, 2018, were enrolled in this study. From this cohort, 22 patients who subsequently experienced spontaneous preterm delivery before 34 weeks of pregnancy formed the study group, and 22 age- and body mass index (BMI)-matched patients without preterm birth constituted the control group. Results No significant differences were observed between the preterm birth and control groups in terms of age, BMI, obstetric history of preterm delivery, gestational age at amniocentesis, or indication for amniocentesis. The mean amniotic fluid levels of ADAMTS4 and ADAMTS5 were significantly increased in the preterm birth group compared to the control group (248.3±22.6 and 182.4±19.8 pg/mL, P=0.012; and 198.6±21.6 and 159.1±21.7 pg/mL, P=0.035, respectively). Significantly increased IL-6 and TNF-α levels were also detected in the amniotic fluid of women who experienced spontaneous preterm delivery, relative to controls (142.1±16.2 and 95.8±16.4 pg/mL, P<0.001; and 139.4±12.5 and 89.6±11.2 pg/mL, P<0.001, respectively). Conclusion The results of this study imply that increased mid-trimester amniotic fluid levels of ADAMTS4, ADAMTS5, IL-6, and TNF-α play an important role in the pathophysiology of spontaneous preterm delivery.

Chondroprotection of PPARα activation by WY14643 via autophagy involving Akt and ERK in LPS-treated mouse chondrocytes and osteoarthritis model.

Autophagy maintains cellular homoeostasis. The enhancement of autophagy in chondrocytes could prevent osteoarthritis (OA) progression in articular cartilage. Peroxisome proliferator‐activated receptor α (PPARα) activation may also protect articular chondrocytes against cartilage degradation in OA. However, whether the protective effect of activated PPARα is associated with autophagy induction in chondrocytes is not determined. In this study, we investigated the effect of PPARα activation by its agonist, WY14643, on the protein expression level of Aggrecan and ADAMTS5, and the protein expression level of autophagy biomarkers, including LC3B and P62, using Western blotting analysis in isolated mouse chondrocytes pre‐treated with lipopolysaccharides (LPS, mimicking OA chondrocytes) with or without the autophagy inhibitor chloroquine diphosphate salt. Furthermore, Akt and ERK phosphorylation was detected in LPS‐treated chondrocytes in response to WY14643. In addition, the effect of intra‐articularly injected WY14643 on articular cartilage in a mouse OA model established by the destabilization of the medial meniscus was assessed using the Osteoarthritis Research Society International (OARSI) histopathology assessment system, along with the detection of Aggrecan, ADAMTS5, LC3B and P62 protein levels using immunohistochemistry assay. The results indicated that PPARα activation by WY14643 promoted proteoglycan synthesis by autophagy enhancement in OA chondrocytes in vivo and in vitro concomitant with the elevation of Akt and ERK phosphorylation. Therefore, autophagy could contribute to the chondroprotection of PPARα activation by WY14643, with the implication that PPARα activation by WY14643 may be a potential approach for OA therapy.

Negative effects of high mechanical tensile strain stimulation on chondrocyte injury in vitro

The mechanism underlying the development of osteoarthritis induced by high tensile strain is unclear. In this study, the effects of different degrees of mechanical tensile strain stimulation on Sprague–Dawley rat chondrocytes were explored. Rat chondrocytes were subjected to mechanical tensile strain at different intensities and frequencies (control group, low tensile strain group, intermediate tensile strain group, and high tensile strain group) using a self-made in vitro tensile strain device. After applying mechanical tensile strain, chondrocytes were collected to detect the expression of collagen II, Aggrecan, matrix metalloproteinase 13 (MMP13), ADAMTS5, and uncoupling protein 2 (UCP2) by real-time quantitative PCR and western blotting as well as reactive oxygen species (ROS) by fluorescence probes. Mechanical tensile strain at different frequencies and intensities had different effects on the biological functions of chondrocytes. Compared with the control group, the expression levels of Col II and Aggrecan in the low and intermediate tensile strain groups increased significantly, while the expression of MMP13 and ADAMTS5 decreased. There were no significant differences between the low and intermediate tensile strain groups. Col II and Aggrecan levels were significantly lower in the high tensile strain group than in the control group, while MMP13 and ADAMTS5 levels were higher. There were no significant differences in ROS production between the low and intermediate tensile strain groups and the control group, but the high tensile strain group exhibited significantly increased ROS production. The expression of UCP2 was significantly lower in the high tensile strain group than in all other groups. These results showed that stimulation with different levels of mechanical tensile strain has different effects on chondrocytes. Repeated high tensile strain promoted the anabolic function of chondrocytes, increased ROS production, and decreased UCP2. These results provide a potential mechanism by which osteoarthritis is induced by high mechanical tensile strain.

The role of ADAMTS genes in the end stage of hip osteoarthritis

PurposeThe aim of this study is to investigate which ADAMTS genes play a major role in the development of primary hip osteoarthritis, by comparing the tissue and blood samples in patients with hip osteoarthritis and a control group. Material and methodsHuman articular cartilage was obtained from femoral heads of 15 patients with end stage osteoarthritis undergoing total hip replacement. As the control group, the cartilages was obtained from femoral heads of 15 patients, who did not have osteoarthritis or degenerative changes in hip joint, undergoing hip replacement following the fracture of the femoral neck. After the cartilage samples were taken from the resection materials, the DNA polymorphisms in the patients' cartilage samples were tested by Polymerase Chain Reaction (PCR), the serum levels of aggrecanase genes were analyzed with Enzyme-Linked ImmunoSorbent Assay (ELISA). ResultsThe level of ADAMTS5 and ADAMTS9 genes were found significantly lower as a result of ELISA analysis degenerative arthritis group than the control group (p < 0,05). ADAMTS 1, 4, 8, 15 were similar between the two groups in ELISA analysis (p > 0,05). As a result of quantitative real time RT-PCR analysis, the level of ADAMTS8 mRNA increased 3.5 fold in hip degenerative arthritis group when compared with femoral neck fractures group. ADAMTS1, ADAMTS4 and ADAMTS5 expression levels in hip degenerative arthritis group were decreased 2.5, 2 and 2.5 fold, respectively. ADAMTS9, 15 were found to be similar between two groups. ConclusonAs a result of this study on hip osteoarthritis, the ADAMTS8 levels was found to be significantly higher in the end stage of hip osteoarthritis. Unlike similar studies on knee osteoarthritis, ADAMTS1,4,5 levels were found to be lower.

Evaluation of second trimester amniotic fluid ADAMTS4, ADAMTS5, interleukin-6 and tumor necrosis factor-α levels in patients with gestational diabetes mellitus.

To test the hypothesis that altered A Disintegrin and Metalloproteinase Domains with Thrombospondins motifs (ADAMTS) is implicated in the etiopathogenesis of gestational diabetes mellitus (GDM). All pregnant women who underwent elective amniocentesis for karyotype analysis between January 1, 2016, and January 1, 2018, were included in this study. From this cohort, the study group consisting of 20 patients diagnosed with GDM was selected and compared against a control group consisting of 20 age- and body mass index (BMI)-matched patients without GDM. ADAMTS4, ADAMTS5, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels were compared in the second trimester amniotic fluid of patients with GDM and normoglycemic pregnant women. No significant differences were observed between GDM and control groups regarding age, BMI, gestational age at amniocentesis and indication for amniocentesis. Mean amniotic fluid ADAMTS4 and ADAMTS5 levels were significantly increased in the GDM group compared with the control group (253.5 ± 18.7 pg/mL and 188.5 ± 21.3 pg/mL, P < 0.001; 192.9 ± 16.4 pg/mL and 154.8 ± 19.9 pg/mL, P = 0.021, respectively). Significant increases in IL-6 and TNF-α levels were also detected in the amniotic fluid of GDM patients relative to controls (136.2 ± 17.3 pg/mL and 98.3 ± 11.5 pg/mL, P < 0.001; 154.2 ± 12.5 pg/mL and 86.2 ± 10.8 pg/mL, P < 0.001, respectively). The data presented here suggest that increased levels of ADAMTS4, ADAMTS5, IL-6 and TNF-α may play an important role in the progression of GDM.