Expression Of ADAMTS-5 Research Articles

ADAMTS4 modulates lung tissue repair following lethal influenza A infection in mice

Abstract Lung tissue damage is a major contributor to morbidity and mortality following infection with influenza A virus. Tissue repair, including remodeling of the extracellular matrix (ECM), is a critical process in recovery from infection. A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family proteins are secreted zinc metalloendopeptidases and important mediators of ECM remodeling. The role of ADAMTS proteins in lung tissue repair following influenza A infection remains unclear. To investigate the regulation of ADAMTS genes in the context of influenza A infection, we performed a microarray analysis of whole lung homogenates harvested at 24 hours post-infection from B6 mice that were uninfected or infected with mouse-adapted influenza A PR8. In the context of infection, several ADAMTS genes, including ADAMTS4, −6, and −9 exhibited a ≥ 3-fold increase in gene expression compared to uninfected lungs. ADAMTS-4 exhibited the most dramatic increase in gene expression (>100-fold) at this early timepoint. We further investigated the role of ADAMTS4 in survival and recovery from influenza A infection by infecting wild-type (WT) and ADAMTS4 −/− knockout B6 mice with a lethal dose of influenza A PR8. Interestingly, ADAMTS4 −/− mice exhibited significantly improved survival compared to WT littermates (log-rank test, p= 0.04) with 75% vs. 41% survival at 15 days post-infection, despite no observable difference in weight loss. Consistent with a role in tissue repair and recovery, differences in survival were observed after 9 days post-infection. These results raise the possibility that increased expression of ADAMTS4 following influenza A infection modulates the tissue repair process and contributes to lung pathology.

In vitro effects of triamcinolone acetonide and in combination with hyaluronan on canine normal and spontaneous osteoarthritis articular cartilage.

The purposes of this study were to examine the cartilage degradation effects of triamcinolone acetonide (TA) on normal and osteoarthritic (OA) primary canine chondrocytes and cartilage explants and to examine the cartilage degradation effects of TA in combination with low-molecular-weight hyaluronan (LMWHA). To assess the effects of these drugs on cell culture, 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and real-time PCR were used to measure chondrotoxicity and determine gene expression, respectively. Uronic acid and hydroxyproline remaining in cartilage and histopathology were used to estimate the effects of these drugs on cartilage explants. In chondrocyte cultures, TA reduced chondrocyte viability in a concentration-dependent manner. LMWHA 2.5mg/ml combined with TA at IC20 (0.09mg/ml) could increase the viability of normal chondrocytes when compared with TA-treated alone. TA at IC20 induced down-regulation of ACAN and induced up-regulation of ADAMTS5 in canine normal chondrocytes. TA at IC20 (0.11mg/ml) up-regulated ADAMTS5, MMP2, MMP3, MMP13, and ACAN expression in canine OA chondrocytes. In explant culture, TA at 1.25, 2.5, and 5mg/ml increased the severity of structural damage, chondrocyte loss and cluster formation, and proteoglycan loss in OA cartilage. LMWHA could decrease the chondrotoxicity of TA at IC20 only in normal chondrocytes, as observed by chondrocyte viability. The combination of LMWHA and TA did not show clearly beneficial effects in all other normal and OA samples. Consequently, using TA alone or in combination with LMWHA in OA cartilage should be of concern because it may lead to cartilage destruction.

Conditional Deletion of Fgfr3 in Chondrocytes leads to Osteoarthritis-like Defects in Temporomandibular Joint of Adult Mice.

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a common degenerative disease in adult, which is characterized by progressive destruction of the articular cartilage. To investigate the role of FGFR3 in the homeostasis of TMJ cartilage during adult stage, we generated Fgfr3f/f; Col2a1-CreERT2 (Fgfr3 cKO) mice, in which Fgfr3 was deleted in chondrocytes at 2 months of age. OA-like defects were observed in Fgfr3 cKO TMJ cartilage. Immunohistochemical staining and quantitative real-time PCR analyses revealed a significant increase in expressions of COL10, MMP13 and AMAMTS5. In addition, there was a sharp increase in chondrocyte apoptosis at the Fgfr3 cKO articular surface, which was accompanied by a down-regulation of lubricin expression. Importantly, the expressions of RUNX2 and Indian hedgehog (IHH) were up-regulated in Fgfr3 cKO TMJ. Primary Fgfr3 cKO chondrocytes were treated with IHH signaling inhibitor, which significantly reduced expressions of Runx2, Col10, Mmp13 and Adamts5. Furthermore, the IHH signaling inhibitor partially alleviated OA-like defects in the TMJ of Fgfr3 cKO mice, including restoration of lubricin expression and improvement of the integrity of the articular surface. In conclusion, our study proposes that FGFR3/IHH signaling pathway plays a critical role in maintaining the homeostasis of TMJ articular cartilage during adult stage.

IL-1β induces low levels of autophagy which clears dysfunctional mitochondria and inhibits ROS, MMP-13 and ADAMTS5 expression in OA chondrocytes

IL-1β induces low levels of autophagy which clears dysfunctional mitochondria and inhibits ROS, MMP-13 and ADAMTS5 expression in OA chondrocytes

IL-6/STAT3 signalling blockade protects against experimental osteoarthritis in mice

IL-6/STAT3 signalling blockade protects against experimental osteoarthritis in mice

Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats

Purpose: Rrecently, we have developed a unilateral anterior crossbite (UAC) procedure that could induce osteoarthritic (OA)-like changes in the temporomandibular joints (TMJs) cartilage of rats and mice. Herein, we employed imaging, histological, and molecular biology techniques, and also in vitra assay, to investigate whether there is mineral deposition in the UAC induced OA cartilage, what the composition the mineral deposition has, and what a role of the minerals may take in OA cartilage. Methods: Spraguee-Dawley rats were subjected to a unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectroscopy (EDS) were used to examine cartilage matrix structures and compositions of mineral deposit in TMJs. Protein and/or RNA expression of modulators of phenotypic markers, mineralization and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic BCP and CPPD crystals were used to stimulate chondrogenic ATDC5 cells for their impact on activities of cellular differentiation and secretion. Results: The proteoglycan content in UAC cartilage began to decrease as early as 2 weeks post-operation and nearly absent at 20 weeks post-UAC without significant inflammatory response. The collagen arrangement was obviously interrupted and the cartialge thickness reduced from 8 weeks but the thickness of the calcified zone was increased. The expression of Col-II, Col-X, and aggrecan was decreased. The control group chondrocytesunder transmission electron microscopy (TEM) were found in well-delimited lacunae that were surrounded by the tightly-organized collagen fibers. In UAC cartilage, however, collagen fibers were disorganized and fragmented with increasing inter-fibers spaces and were worsened with time. Mineral deposition was observed around the hypertrophic chondrocytes, beginning 2 weeks after the UAC procedure. Those peri-cellular minerals were contained in abundant matrix vesicles (with diameters ranging from 50-200 nm) that were located in close proximity to the cell member of hypertrophic chondrocytes, supporting the cellular origin of the vesicles. Immunohistochemistry and/or real-time PCR analyses demonstrated biphasic changes in the expression of MGP mRNA in TMJ joints of UAC rat, which increased at 2 and 4 weeks post-operation and decreased after 8 weeks post-operation when compared to age-matched controls. The expression of CD73 and Ank was significantly reduced in TMJ cartilage of UAC group at all time points. The expression of Npp1 was significantly reduced in UAC cartilage at 2 and 4 weeks post-operation. In contrast to the expression of anti-mineralization molecules, the expression of Tnap, which hydrolyzes PPi into Pi to promote the formation of BCP crystals, was up-regulated in the UAC groups at 4, 8, and 12 weeks post-operation, and this effect waned at 20 weeks post-operation. The expression of matrix metalloproteinase, Mmp13, which cleaves type II collagen, was also up-regulated in the UAC groups in the similar time-dependent manner as seen in the expression of Tnap. Using EDS technology we confirmed an increase in calcium (Ca) and phosphorus (P) content in UAC cartilage vs their age-matched controls, beginning at 2 weeks post-operation. Only one of four joints tested in 12- and 20-week UAC group was noticed an isolated mineral which was located closing to the deep zonal mineral deposition. They were rich in calcium and phosphorus, but had a low carbon content. Their averaged Ca/P ratio was 1.42 (ranging from 1.38 to 1.48) and 1.44 (range from 1.39 to 1.49) at 12- and 20-weeks UAC group, respectively, closely resemble to those of octacalcium phosphate (Ca/P=1.33) and tricalcium phosphate (Ca/P=1.50), two subtypes of BCP crystals. We then exposed ATDC5 cells to the synthetic BCP crystals. We found that BCP crystals (50μg/ml) reduced Col-II, Col-X and aggrecan expression, but upregulated the expression of Adamts5, Mmp3 and Mmp9, however, only transiently stimulated Tnf-α and Il-1β expression. Conclusions: The present data indicated that mechanical stimuli by UAC enhance mineral accumulation of BCP-like minerals in the cartilage matrix of TMJ condyle at deep layer. The UAC induced aberrant mineralization deposition of BCP-like minerals can stimulate matrix degradation via promoting cartilage-degrading enzymes, and may contribute, at least in part, to the thinning of articular cartilage by expansion of the calcified cartilage, facilitate osteoarthritis progression.

RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.

Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1β (IL-1β), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1β activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1β by RNA interference (RNAi) may drive a reduction in IL-1β. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1β (shIL-1β) would significantly decrease IL-1β expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1β in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1β knockdown led to significantly decreased expression of IL-1β, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1β using rAAV2 vector expressing a short hairpin IL-1β knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints.

Protective effect of LR-90 on articular cartilage in rabbit model of osteoarthritis

To investigate the protective effect of LR-90 on articular cartilage in rabbit model of osteoarthritis. The cultured rabbits chondrocytes were assigned to be treated with IL-1β (10ng/ml) or IL-1β (10ng/ml)+LR-90 (50 mg/L). The mRNA expression of MMP-13, ADAMTS-5, aggrecan and collagen II in chondrocytes were assessed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Twenty male New Zealand white rabbits underwent bilateral anterior cruciate ligament transection (ACLT) to establish a animal model of osteoarthritis. Four weeks after model established, on the basis of randomization one knee of each rabbit was treated with 50 mg/L LR-90 in normal saline (NS) (experimental group) and the other knee was treated with same volume of NS (control group), 1/week × 5. Nine weeks after ACLT all rabbits were sacrificed and the knee joints were evaluated by gross morphology and histology. The mRNA expression of IL-1β, MMP-13, ADAMTS-5, aggrecan and collagen Ⅱ in articular cartilage was analyzed by RT-PCR. Gross morphology and Mankin histological evaluation showed that the extent and grade of cartilage damage in the experimental group were less severe than those in the control group.Compared to IL-1β group, LR-90 treatment suppressed the mRNA expression of MMP-13 and ADAMTS-5, and enhanced aggrecan and collagen Ⅱ mRNA expression. Consistent with the in vitro results, the intraarticular LR-90 administration suppressed the mRNA expression of IL-1β,MMP-13 and ADAMTS-5 (all P<0.01), while enhanced mRNA expression of aggrecan and collagen Ⅱ in cartilage (all P<0.01). LR-90 protects against cartilage degradation and inhibits the progression of osteoarthritis in rabbit mode1 of osteoarthritis, which is associated with the suppressing IL-1β, MMP-13, ADAMTS-5 and promoting aggrecan and collagen Ⅱ mRNA expression in cartilage.

Versican: a novel modulator of hepatic fibrosis

Little is known about the deposition and turnover of proteoglycans in liver fibrosis, despite their abundance in the extracellular matrix. Versican plays diverse roles in modulating cell behavior in other fibroproliferative diseases, but remains poorly described in the liver. Hepatic fibrosis was induced by carbon tetrachloride treatment of C57BL/6 mice over 4 weeks followed by recovery over a 28-day period. Primary mouse hepatic stellate cells (HSCs) were activated in culture and versican was transiently knocked down in human (LX2) and mouse HSCs. Expression of versican, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs (ADAMTS)-1, -4, -5, -8, -9, -15, and -20, and markers of fibrogenesis were studied using immunohistochemistry, real-time quantitative PCR, and western blotting. Immunohistochemistry showed increased expression of versican in cirrhotic human livers and the mouse model of fibrosis. Carbon tetrachloride treatment led to significant increases in versican expression and the proteoglycanases ADAMTS-5, -9, -15, and -20, alongside TNF-α, α-smooth muscle actin (α-SMA), collagen-1, and TGF-β expression. During recovery, expression of many of these genes returned to control levels. However, expression of ADAMTS-5, -8, -9, and -15 showed delayed increases in expression at 28 days of recovery, which corresponded with decreases in versican V0 and V1 cleavage products (G1-DPEAAE1401 and G1-DPEAAE441). Activation of primary HSCs in vitro significantly increased versican, α-SMA, and collagen-1 expression. Transient knockdown of versican in HSCs led to decreases in markers of fibrogenesis and reduced cell proliferation, without inducing apoptosis. Versican expression increases during HSC activation and liver fibrosis, and proteolytic processing occurs during the resolution of fibrosis. Knockdown studies in vitro suggest a possible role of versican in modulating hepatic fibrogenesis.

Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

We examined whether apigenin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effects of apigenin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription - polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of apigenin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of apigenin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, apigenin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Furthermore, apigenin inhibited the secretion and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that apigenin can regulate the gene expression, secretion, and activity of MMP-3, by directly acting on articular chondrocytes.

Fluid-induced, shear stress-regulated extracellular matrix and matrix metalloproteinase genes expression on human annulus fibrosus cells.

BackgroundMechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device.MethodshAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm2 shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression.ResultsShear stress of 1 dyne/cm2 exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm2 had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm2 group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm2 groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups.ConclusionTaken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.

Differential expression of p38 MAPK α, β, γ, δ isoforms in nucleus pulposus modulates macrophage polarization in intervertebral disc degeneration.

P38MAPK mediates cytokine induced inflammation in nucleus pulposus (NP) cells and involves in multiple cellular processes which are related to intervertebral disc degeneration (IDD). The aim of this study was to investigate the expression, activation and function of p38 MAPK isoforms (α,β, γ and δ) in degenerative NP and the effect of p38 activation in NP cells on macrophage polarization. P38 α, β and δ isoforms are preferential expressed, whereas the p38γ isoform is absent in human NP tissue. LV-sh-p38α, sh-p38β transfection in NP cells significantly decreased the ADAMTS-4,-5, MMP-13,CCL3 expression and restored collagen-II and aggrecan expression upon IL-1β stimulation. As compared with p38α and p38β, p38δ exhibited an opposite effect on ADAMTS-4,-5, MMP-13 and aggrecan expression in NP cells. Furthermore, the production of GM-CSF and IFNγ which were trigged by p38α or p38β in NP cells induced macrophage polarization into M1 phenotype. Our finding indicates that p38 MAPK α, β and δ isoform are predominantly expressed and activated in IDD. P38 positive NP cells modulate macrophage polarization through the production of GM-CSF and IFNγ. Hence, Our study suggests that selectively targeting p38 isoforms could ameliorate the inflammation in IDD and regard IDD progression.

Role of ADAMTS1 and ADAMTS5 in male infertility.

The aim of this study was to investigate the relationship of infertility with metalloproteinases ADAMTS1 and ADAMTS5, which are known to be responsible for the degradation of extracellular matrix (ECM) proteins associated with many diseases. ECM is the noncellular component that provides structural and biochemical support to the surrounding cells required for tissue morphogenesis, differentiation and homoeostasis. Sixty infertile individuals and 10 healthy semen donors were included in this study. The infertile individuals were classified as normozoospermia (NS; n=20), oligozoospermia (OS; n=20), azoospermia (AS; n=20) groups. ADAMTS1 and ADAMTS5 protein levels in semen were analysed by Western blot. ADAMTS1 protein level was 3.0-, 3.3- and 1.6-fold lower in the OS, AS and NS groups, respectively, than in the control group (P<0.001). ADAMTS5 protein level was 3.2-, 2.7- and 1.4-fold lower in the OS, AS and NS groups, respectively, than in the control group (P<0.001). Sperm count and sperm motility showed a negative correlation with the levels of ADAMTS1 and ADAMTS5 protein expression: r=-0.477, r=-0.470; and r=-0.332, r=-0.275 respectively (P<0.001). In conclusion, ADAMTS1 and ADAMTS5 protein expressions in semen are significantly related with sperm production. It is very important to understand molecular function and organisation of ADAMTSs which will be significant in enlightening the process of spermatogenesis in male infertility.

Parathyroid hormone(1-34) exhibits more comprehensive effects than celecoxib in cartilage metabolism and maintaining subchondral bone micro-architecture in meniscectomized guinea pigs

To evaluate the effects of PTH(1-34) on cartilage, subchondral bone mass and structure in medial meniscectomized guinea pigs and compare them to those of celecoxib (CLX). Forty-eight 3-month-old male Hartley albino guinea pigs received either sham or medial meniscectomy (MNX) operations. One week after the procedure, meniscectomized animals began 12 weeks of treatment by oral administration of CLX (20mg/kg, daily), subcutaneous injection of PTH (1-34) (24μg/kg, 5 days/week), or normal saline for MNX group. All animals were euthanized 12 weeks later, cartilage degeneration and subchondral bone micro-architecture was analyzed. OARSI scores indicated cartilage degeneration was partially inhibited by either CLX or PTH(1-34). Cartilage was significantly thicker in PTH(1-34)-treated animals than in CLX-treated animals. Both CLX and PTH(1-34) treatment were associated with lower ADAMTS-4 and periostin expression than MNX. MMP-13 expression in PTH(1-34) group was significantly lower than that in CLX group. However, AGG expression and the ratio of Col-II/MMP-13 expression in PTH(1-34) group were significantly higher than in the CLX group. Micro-CT analysis showed BMD, BV/TV, and Tb.Th levels to be significantly lower in the MNX group and CLX groups than in the sham group, but these parameters were significantly higher in the PTH(1-34) group than in either the MNX group or CLX group. Both CLX and PTH(1-34) exhibits protective effects on cartilage degeneration in meniscectomized guinea pigs. However, PTH(1-34) exhibited superior performance to CLX not only in metabolism of cartilage tissue but also in maintenance of subchondral bone micro-architecture.

Coordinated regulation of mirnas to induce chondrogenesis and inhibit angiogenesis in stem cells and ECM materials under hypoxic environment

Event Abstract Back to Event Coordinated regulation of mirnas to induce chondrogenesis and inhibit angiogenesis in stem cells and ECM materials under hypoxic environment Manman Gao1, Shaoming Chen1, Zhiyu Zhou1, Jiabi Liang1, Ming Gong1, Xuejun Dai1, Tangzhao Liang1, Gang Wu1 and Xuenong Zou1 1 The First Affiliated Hospital of Sun Yat-sen University, Department of Spine Surgery, Orthopaedic Research Center, China Background: Currently, cartilage repair biomaterials loaded with or without cytokines have been widely studied. But few have achieved complete and stable cartilage regeneration. One of the crucial reasons is that the mesenchymal stem cells (MSCs) seeded in cartilage repair biomaterials would be affected in the microenvironment under hypoxia. Hypoxia would potentially induce angiogenesis, resulting cartilage secondary degeneration. To explore the antagonism of chondrogenesis and angiogenesis, we investigated the coordinated regulation of epigenetic mechanisms in MSCs and ECM materials under hypoxic environment. The present study is aimed to guide the design and synthesis of biological adaptive cartilage repair biomaterials. Materials and Methods: C3H10T1/2 cell line was used in this study. C3H10T1/2 cell line seeded as pellet or in ECM materials was added chondrogenic medium (with 10 ng/mL TGFβ3) or DMEM medium for 21 days under hypoxia (2% O2) or normoxia (21% O2). Genes and miRNAs related with chondrogenesis and angiogenesis were detected by RT-qPCR technique on Day 7, 14, 21. Dual-luciferase report system was used to explore the regulating mechanisms of miRNAs on angiogenesis. Result: Pellet culture showed higher expression of Sox9, Col2a1, Acan, and lower expression of Mmp13 and Adamts5 with chondrogenic medium than with none chondrogenic medium (P<0.05), which indicated chondrogenesis of C3H10T1/2 cell line. As previously reported, HIF1α was up-regulated under hypoxia environment compared with normoxia environment (P<0.05). Meanwhile, Sox9, Col2a1 and Acan were higher expressed and Adamts5 and Mmp13 were lower expressed (P<0.05). As a key regulator, miR-140 exhibited higher expression and miR-146 lower expression. ECM materials were mainly composed of hyaluronic acid and chondroitin sulfate crosslinking the form of hydrogel. Hoechst 33258 fluorescent staining validated its cytocompatibility with few dead cells and immunohistochemical staining showed abundant Collagen type Ⅱ on Day 21 in the chondrogenic induction of C3H10T1/2 cells. The chondrogenic phenotype was more stabilized in the hydrogel of ECM materials with chondrogenic medium than DMEM medium, showing higher Sox9, Col2a1, Acan while lower Adamts5, Mmp13 expressed (P<0.05). At the same time, miR-140 was up-expressed and miR-146 down-expressed. Dual-luciferase report assays demonstrated that miR-140 directly targets VEGFα by binding its 3'-UTR. Conclusions: Bio-adaptation of chondrogenesis and anti-angiogenesis would be coordinated by means of regulation of MSCs differentiation with biological active factors, ECM materials and hypoxia. Up-regulation of miR-140 and down-regulation of miR-146 probably played important regulating effects on promoting chondrogenesis as well as inhibiting angiogenesis. Bioactive modification with these regulatory molecules would enhance the bio-adaptation of cartilage repair materials. National Basic Research Program of China (973 Program, No. 2012CB619100); National Nature Science Fund of China (No. 31430030, 0731001, 81071512)

ADAMTS5 promotes murine adipogenesis and visceral adipose tissue expansion.

Enhanced expression of the aggrecanase ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) has been observed in adipose tissue (AT) of obese rodents. Here, we have investigated the role of ADAMTS5 in adipogenesis, AT expansion and associated angiogenesis. In vitro differentiation of precursor cells into mature adipocytes was studied using murine embryonic fibroblasts (MEF) derived from wild-type (Adamts5(+/+)) and ADAMTS5 deficient (Adamts5(-/-)) mice, or 3T3-F442A preadipocytes with stable gene silencing of Adamts5. De novo adipogenesis was monitored by injection of 3T3-F442A cells with or without Adamts5 knockdown in Nude mice. Furthermore, Adamts5(+/+)and Adamts5(-/-) mice were kept on a high-fat diet (HFD) to monitor AT development. Adamts5(-/-) MEF, as well as 3T3-F442A preadipocytes with Adamts5 knockdown, showed significantly reduced differentiation as compared to control cells. In mice, de novo formed fat pads arising from 3T3-F442A cells with Adamts5 knockdown were significantly smaller as compared to controls. After 15 or 25 weeks on HFD, total body weight and subcutaneous AT weight were similar for Adamts5(+/+) and Adamts5(-/-) mice, but visceral/gonadal fat mass was significantly lower for Adamts5(-/-) mice. These data were confirmed by magnetic resonance imaging. In addition, the blood vessel density in adipose tissue was higher for Adamts5(-/-) mice kept on HFD. In conclusion, our data support the concept that ADAMTS5 promotes adipogenesis in vitro and in vivo, as well as development of visceral AT and associated angiogenesis in mice kept on HFD.

Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo.

We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Inhibition of Osteoarthritis in Rats by Electroporation with Interleukin-1 Receptor Antagonist

Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated virus (AAV) vectors. EP- and AAV-mediated delivery of human interleukin-1 receptor antagonist (hIL-1Ra) was localized performed in the joints of rats following induction of OA. mRNA levels for hIL-1Ra, IL-1β, TNF-α, MMP-13 and ADAMTS-4 in the cartilage and synovial tissues were analyzed. Structural analyses of the subchondral bone at the medial femoral condyle were performed by Micro-CT after treatment. Knee joint specimens were staining with hematoxylin and eosin and Saffron O. Induction of hIL-1Ra by both EP and AAV inhibited inflammatory-induced sub-chondral bone reconstruction, and effectively suppressed IL-1β activity, as evidenced by decreased expression of MMP-13 and ADAMTS-4. Histological analyses revealed significant protection of cartilage, proteoglycan by EP and AAV. hIL-1Ra expression was similar in both the EP and AAV groups. Notably, this gene is not easier degraded transduced by EP compared with AAV. Taken together, these results show that EP offers transfection efficiency comparable to that of AAV, with the potential for longer gene expression, making EP a promising candidate for efficient non-viral delivery of OA gene therapy.

The Investigation of a Disintegrin and Metalloproteinase with ThromboSpondin Motifs (ADAMTS) 1, 5 and 16 in Thoracic Aortic Aneurysms and Dissections

Disintegrin-like and Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins that are fundamentally located in the extracellular matrix (ECM) have critical roles on different cellular processes by altering the ECM architecture. It has been known that expression of some members of these proteinases increases in aneurismal and dissectional aortic tissue. The purpose of this study is to investigate ADAMTS1, 5, 16 and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) levels in aortic tissue obtained from patients with thoracic aortic aneurysms and dissections and to achieve new insights about the function of ADAMTS family members. We investigated ADAMTS1, 5, and 16 expression in human thoracic aortic aneurysms (TAA) (n = 22), thoracic aortic dissections (TAD) (n = 12), and thoracic aortas from age-matched control organ donors (n = 6) (a total number of 34 cases and 6 controls). The expression levels of ADAMTS proteins were determined by Western blot technique using anti-ADAMTS1, ADAMTS5, ADAMTS16, TIMP-1 and TIMP-2 antibodies. ADAMTS1, 5, and 16 protein expressions were significantly higher in thoracic aortic aneurysm and dissection tissues compared to control aortic tissues. Furthermore, TIMP-1 protein levels decreased in TAA and TAD tissues, TIMP-2 did not change. Under the light of our findings, increased expression of ADAMTS1, 5, and 16 proteins may promote deceleration in thoracic aortic aneurysm progression. This is the first study that demonstrates ADAMTS5 and ADAMTS16 proteolytic activity in aneurysm and dissection.

Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats

To investigate whether mechanical stress induces mineral deposits that contribute to matrix degradation at the onset of osteoarthritis (OA) in temporomandibular joint (TMJ) cartilage. Female Spraguee-Dawley rats were subjected to an unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectrometer (EDS) were used to examine cartilage matrix structures and composition of mineral deposit in the affected TMJ cartilage. Protein and/or RNA expression of phenotypic markers and mineralization modulators and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic basic calcium phosphate (BCP) and calcium pyrophosphate dehydrate (CPPD) crystals were used to stimulate ATDC5 cells for their impact on cell differentiation and gene expression. Fragmented and disorganized collagen fibers, expanded fibrous spaces, and enhancement of matrix vesicle production and mineral deposition were observed in matrices surrounding hypertrophic chondrocytes in cartilage as early as 2-weeks post-UAC and exacerbated with time. The mineral deposits in TMJ cartilage at 12- and 20-weeks post-UAC had Ca/P ratios of 1.42 and 1.44, which are similar to the ratios for BCP. The expression of mineralization inhibitors, NPP1, ANK, CD73, and Matrix gla protein (MGP) was decreased from 2 to 8 weeks post-UAC, so were the chondrogenic markers, Col-2, Col-X and aggrecan. In contrast, the expression of tissue-nonspecific alkaline phosphatase (TNAP) and MMP13 was increased 4-weeks post-UAC. Treating ADTC5 cells with BCP crystals increased MMPs and ADAMTS5 expression, but reduced matrix production in a time-dependent manner. UAC induces deposition of BCP-like minerals in osteoarthritic cartilage, which can stimulate matrix degradation by promoting the expression of cartilage-degrading enzymes to facilitate OA progression.