Expression Of Matrix Metallopeptidase Research Articles

MiR-223 promotes cartilage differentiation of bone marrow-derived mesenchymal stem cells and protects against osteoarthritis by suppressing NLRP-3 expression

IntroductionThe present investigation evaluates the role of miR-223 mimic in the treatment of osteoarthritis (OA) and postulates the possible molecular mechanism of its action.Material and methodsBone marrow-derived mesenchymal stem cells (BMSCs) were isolated from rats and cultured in chondrogenic medium to stimulate the differentiation of chondrocytes. Alcian blue staining was performed to determine the chondrogenic differentiation and expression of miR-223 in the BMSCs. Moreover, expression of NLR family pyrin domain containing 3 (NLRP-3), matrix metallopeptidase-13 (MMP-13) and collagen (Col II) was determined in miR-223 mimic and inhibitor treated BMSCs. OA was induced by injecting anterior cruciate ligament transection in rats followed by further treatment with the miR-223 mimic for the period of the treatment protocol. Level and expression of inflammatory cytokines were estimated in the cartilage tissue of OA rats. Moreover, immunohistochemical analysis and histopathology study were also performed.ResultsData of the study reveal that expression of miR-223 was higher in chondrogenic differentiated BMSCs than normal. Expression of MMP-13 and NLRP-3 was lower, and expression of Col II was higher in miR-223 mimic treated BMSCs than normal. Moreover, data of the in-vivo study indicate that the expression level of cytokines was lower in the cartilage tissue of the miR-223 mimic treated group than the OA group. Treatment with the miR-223 mimic ameliorates the altered histopathology and expression of NLRP-3 in the cartilage tissue of OA rats.ConclusionsData of the study reveal that the miR-223 mimic enhances the chondrogenic differentiation of BMSCs by regulating the NLRP-3/IL-18/TGF-b pathway.

Pharmaco-molecular assessment of the effects of anandamide and its antagonists on hippocampal tissue in Wistar albino rats.

The members of the matrix metalloproteinase (MMP) family and cannabinoids (CBs) are reportedly associated with hippocampus-dependent memory functions. However, the effects of endogenously formed CBs on hippocampal long-term potentiation remain unknown. The present study aimed to investigate the changes in the gene and protein expression levels of matrix metallopeptidase 9 (MMP-9), phosphatase and tensin homolog (PTEN), and NOTCH receptor 1 (NOTCH1) in rat hippocampal tissues treated with anandamide (AEA), AM251, 6-iodopravadolin (AM630), and N-[4-{[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl}phenyl] (ML193). The subjects were divided into 10 groups (n = five per group). The pharmaceuticals were administered via intraperitoneal injection once a day for seven days, except for the control group. The resected hippocampal tissues were then evaluated using a quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. The data obtained were statistically analyzed, and p < 0.01 was considered statistically significant. Contrary to the literature, the changes in MMP-9 expression were not statistically significant, but the changes in PTEN and NOTCH1 were. The findings of this in vivo experimental study revealed that the agonists and antagonists acting on the CB system have significant molecular effects on hippocampal tissue. The changes in gene and protein expressions may be one of the reasons for the neurodegenerative processes observed in patients using these agonists and antagonists, whose effects on the CB system have not been fully explained yet. Our study can contribute to the literature as it is the first study investigating the MMP-9, PTEN and NOTCH1 gene and protein expression.

Inhibition of hypertrophy and improving chondrocyte differentiation by MMP-13 inhibitor small molecule encapsulated in alginate-chondroitin sulfate-platelet lysate hydrogel

BackgroundMesenchymal stem cells are a promising cell source for chondrogenic differentiation and have been widely used in several preclinical and clinical studies. However, they are prone to an unwanted differentiation process towards hypertrophy that limits their therapeutic efficacy. Matrix metallopeptidase 13 (MMP-13) is a well-known factor regulated during this undesirable event. MMP-13 is a collagen degrading enzyme, which is also highly expressed in the hypertrophic zone of the growth plate and in OA cartilage. Accordingly, we investigated the effect of MMP-13 inhibition on MSC hypertrophy.MethodsIn this study, 5-bromoindole-2-carboxylic acid (BICA) was used as an inhibitory agent for MMP-13 expression. After identifying its optimal concentration, BICA was mixed into a hydrogel and the release rate was studied. To prepare the ideal hydrogel, chondroitin sulfate (CS) and platelet lysate (PL) were mixed with sodium alginate (Alg) at concentrations selected based on synergistic mechanical and rheometric properties. Then, four hydrogels were prepared by combining alginate (1.5%w/v) and/or CS (1%w/v) and/or PL (20%v/v). The chondrogenic potential and progression to hypertrophy of human bone marrow-derived mesenchymal stem cell (hBM-MSC)-loaded hydrogels were investigated under free swelling and mechanical loading conditions, in the presence and absence of BICA.ResultsViability of hBM-MSCs seeded in the four hydrogels was similar. qRT-PCR revealed that BICA could successfully inhibit MMP-13 expression, which led to an inhibition of Coll X and induction of Coll-II, in both free swelling and loading conditions. The GAG deposition was higher in the group combining BICA and mechanical stimulation.ConclusionsIt is concluded that BICA inhibition of MMP-13 reduces MSC hypertrophy during chondrogenesis.Graphical abstract

Effects of mild steel welding fume particles on pulmonary epithelial inflammation and endothelial activation

Welders have an increased risk for cardiovascular disease (CVD) following exposure to welding fumes. The underlying mechanisms are largely unknown; however, oxidative stress, systemic inflammation, and endothelial dysfunction have been suggested as contributing factors to particle-induced CVD. We investigated effects of mild steel welding fume (MSWF) on three target cell types: macrophages, pulmonary epithelial, and vascular endothelial cells. Cells were exposed to MSWF at nontoxic doses for 6 h/day, for five consecutive days. The expression of 40 genes involved in inflammation, fibrosis, and endothelial activation was analyzed. Moreover, changes in the reactive oxygen species production and migration capacity of cells were assessed. The expression of matrix metallopeptidase 1 (MMP1) was induced in both epithelial and endothelial cells following repeated exposure to MSWF. Although MMP1 is important in inflammatory responses in vivo, this effect was not concurrent with changes in the inflammatory status, cell proliferation, and migration capacities, nor did it induce oxidative stress in the cells. Thus, repeated exposure with low doses of MSWF was sufficient neither for inducing inflammatory stress in epithelial cells and macrophages nor for endothelial activation, and higher concentrations of MSWF or the nonparticle fraction of MSWF may be critical in causing the increased risk of CVD observed among welders.

Primary Cilia in Trophoblastic Cells: Potential Involvement in Preeclampsia.

The pathogenesis of preeclampsia, a pregnancy-related disease, is not completely understood. The primary cilium transduces a diverse array of signaling pathways important for vital cellular activities. Primary cilia were reported to facilitate trophoblastic cell invasion. We hypothesized their further functions in trophoblasts and were interested in related molecular mechanisms. We systematically examined the presence, length and percentage of the primary cilium, its mediated signal transduction, and its connection to trophoblast function. Various cellular and molecular methods were used including immunofluorescence staining, spheroid formation, gene analysis, invasion and tube formation assays with trophoblastic cell lines, primary trophoblasts, and placental tissues. We show that primary cilia are present in various trophoblastic cell lines derived from first trimester placentas. Cilia are also observable in primary trophoblasts, though in a small quantity. Importantly, primary cilia are shortened in trophoblastic cells derived from preeclamptic placentas. Mechanistically, interleukin-6, tumor necrosis factor-α or sera from patients with preeclampsia are able to reduce the length of primary cilia and impair the important sonic hedgehog signaling pathway. Functionally, trophoblastic cells with defective cilia display severe failures in their key functions, like migration, invasion and tube formation, also observed in trophoblastic cells depleted of the intraflagellar transport protein 88. This is accompanied by reduced gene expression of matrix metallopeptidases, vascular endothelial growth factor, and placental growth factor. This work highlights the significance of primary cilia in the functions of trophoblastic cells. Dysfunctional cilia may lead to compromised migration, invasion, and endothelial remodeling of trophoblastic cells, contributing to the development of preeclampsia.

Thrombospondin-2 stimulates MMP-9 production and promotes osteosarcoma metastasis via the PLC, PKC, c-Src and NF-κB activation.

Osteosarcoma is an extremely common primary bone malignancy that is highly metastatic, with most deaths resulting from pulmonary metastases. The extracellular matrix protein thrombospondin‐2 (TSP‐2) is key to many biological processes, such as inflammation, wound repair and tissue remodelling. However, it is unclear as to what biological role TSP‐2 plays in human metastatic osteosarcoma. The immunochemistry analysis from osteosarcoma specimens identified marked up‐regulation of TSP‐2 in late‐stage osteosarcoma. Furthermore, we found that TSP‐2 increased the levels of matrix metallopeptidase 9 (MMP‐9) expression and thereby increased the migratory potential of human osteosarcoma cells. Osteosarcoma cells pre‐treated with an MMP‐9 monoclonal antibody (mAb), an MMP‐9 inhibitor, or transfected with MMP‐9 small interfering RNA (siRNA) reduced the capacity of TSP‐2 to potentiate cell migration. TSP‐2 treatment activated the PLCβ, PKCα, c‐Src and nuclear kappa factor B (NF‐κB) signalling pathways, while the specific siRNA, inhibitors and mutants of these cascades reduced TSP‐2‐induced stimulation of migration activity. Knockdown of TSP‐2 expression markedly reduced cell metastasis in cellular and animal experiments. It appears that an interaction between TSP‐2 and integrin αvβ3 activates the PLCβ, PKCα and c‐Src signalling pathways and subsequently activates NF‐κB signalling, increasing MMP‐9 expression and stimulating migratory activity amongst human osteosarcoma cells.

Dexamethasone downregulates the expressions of MMP-9 and oxidative stress in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

Steroids have been shown to be beneficial in patients and mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection; however, the mechanism for this beneficial effect is unknown. We speculated that the effect of steroids in eosinophilic meningitis caused by A. cantonensis infection may be mediated by the downregulation of matrix metallopeptidase-9 (MMP-9) and oxidative stress pathways via glucocorticoid receptors (GRs). We found blood-brain barrier (BBB) dysfunction in mice with eosinophilic meningitis 2-3 weeks after infection as evidenced by increased extravasation of Evans blue and cerebrospinal fluid (CSF) albumin levels. The administration of dexamethasone significantly decreased the amount of Evans blue and CSF albumin. The effect of dexamethasone was mediated by GRs and heat shock protein 70, resulting in subsequent decreases in the expressions of nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in the CSF and brain parenchymal after 2 weeks of steroid administration. Steroid treatment also decreased CSF/brain homogenate MMP-9 concentrations, but had no effect on CSF MMP-2 levels, indicating that MMP-9 rather than MMP-2 played a major role in BBB dysfunction in mice with eosinophilic meningitis. The concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) gradually increased after 1-3 weeks of infection, and the administration of dexamethasone significantly downregulated the concentration of oxidized derivative 8-OHdG in CSF. In conclusion, increased 8-OHdG and MMP-9 concentrations were found in mice with eosinophilic meningitis caused by A. cantonensis infection. The effect of dexamethasone was mediated by GRs and significantly decreased not only the levels of 8-OHdG and MMP-9 but also NF-κB, JNK and ERK.

Puerarin inhibits titanium particle-induced osteolysis and RANKL-induced osteoclastogenesis via suppression of the NF-κB signaling pathway.

Osteolysis around the prosthesis and subsequent aseptic loosening are the main causes of prosthesis failure. Inflammation due to wear particles and osteoclast activation are the key factors in osteolysis and are also potential targets for the treatment of osteolysis. However, it is not clear whether puerarin can inhibit chronic inflammation and alleviate osteolysis. In this study, we investigated the effect of puerarin on Ti particle‐induced inflammatory osteolysis in vivo in rat femoral models and in vitro in receptor activator of nuclear factor kappa‐B ligand (RANKL)‐induced osteoclast activation models. Our in vivo results showed that puerarin significantly inhibited Ti particle‐induced osteolysis and the expression of matrix metallopeptidase 9 (MMP‐9), nuclear factor of activated T cells 1 (NFATc1), tumour necrosis factor (TNF)‐α and interleukin (IL)‐6. In vitro, puerarin prevented RANKL‐induced osteoclast differentiation, bone resorption and F‐actin ring formation in a concentration‐dependent manner. Furthermore, puerarin decreased the phosphorylation of p65 and prevented p65 moving from the cytoplasm to the nucleus. Puerarin also reduced the expression of osteoclast‐specific factors and inhibited the inflammatory response. In conclusion, our study proves that puerarin can block the NF‐κB signalling pathway to inhibit osteoclast activation and inflammatory processes, which provides a new direction for the treatment of osteolysis‐related diseases.

A novel treatment for skin repair using a combination of spironolactone and vitamin D3

Injury of the skin from exposure to toxic chemicals leads to the release of inflammatory mediators and the recruitment of immune cells. Nitrogen mustard (NM) and other alkylating agents cause severe cutaneous damage for which there are limited treatment options. Here, we show that combined treatment of vitamin D3 (VD3) and spironolactone (SP), a mineralocorticoid receptor antagonist, significantly improves the resolution of inflammation and accelerates wound healing after NM exposure. SP enhanced the inhibitory effect of VD3 on nuclear factor‐kB activity. Combined treatment of NM‐exposed mice with VD3 and SP synergistically inhibited the expression of iNOS in the skin and decreased the expression of matrix metallopeptidase‐9, C‐C motif chemokine ligand 2, interleukin (IL)‐1α, and IL‐1β. The combined treatment decreased the number of local proinflammatory M1 macrophages resulting in an increase in the M2/M1 ratio in the wound microenvironment. Apoptosis was also decreased in the skin after combined treatment. Together, this creates a proresolution state, resulting in more rapid wound closure. Combined VD3 and SP treatment is effective in modulating the immune response and activating anti‐inflammatory pathways in macrophages to facilitate tissue repair. Altogether, these data demonstrate that VD3 and SP may constitute an effective treatment regimen to improve wound healing after NM or other skin chemical injury.

CEACAM1 Inhibited IκB-α/NF-κB Signal Pathway Via Targeting MMP-9/TIMP-1 Axis in Diabetic Atherosclerosis.

Atherosclerosis (AS) is the most common and serious complication in type 2 diabetes mellitus (T2DM). Recent studies have emphasized that inflammation is the main cause of atherosclerosis. Studies have shown that carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) regulates the expression of matrix metallopeptidase 9 (MMP-9) after ischemic stroke to reduce inflammation. The aim of this study was to elucidate potential molecular mechanism of CEACAM1 on the inflammatory response in atherosclerosis. The serum levels of CEACAM1, MMP-9, and tissue inhibitors of metalloproteinase 1 (TIMP-1) in T2DM patients and healthy control was detected. The results showed that the levels of CEACAM1 and TIMP-1 were significantly decreased, and the levels of MMP-9 were significantly higher than those in the control group. Moreover, we also observed the effect of CEACAM1 on atherosclerosis in T2DM rats. Hematoxylin & eosin (HE) staining and oil red staining showed that CEACAM1 recombinant protein reduced intima-media thickness and the area of atherosclerotic plaques. To further explore the molecular mechanism of CEACAM1 regulating MMP-9/TIMP-1, we conducted experiments in rat aorta vascular endothelial cells and rat aorta smooth muscle cells. The result showed that CEACAM1 inhibits inflammatory response via MMP-9/TIMP-1 axis. Taken together, CEACAM1 attenuates diabetic atherosclerosis by inhibition of IκB/NF-κB signal pathway via MMP-9/TIMP-1 axis, which indicate that CEACAM1 is potentially amenable to therapeutic manipulation for clinical application in atherosclerosis in T2DM.

Ceramide induces MMP-9 expression through JAK2/STAT3 pathway in airway epithelium

BackgroundCeramide, a bioactive lipid, plays an essential role in the development of several pulmonary inflammatory diseases. Matrix metallopeptidase 9 (MMP-9) regulates the synthesis and degradation of extracellular matrix, and is associated with airway remodeling and tissue injury. This study was conducted to investigate the effects and underlying mechanisms of ceramide on MMP-9 expression in airway epithelium.MethodsBEAS-2B cells, normal human bronchial epithelium cell lines, were pretreated with AG490, a selective janus tyrosine kinase 2 (JAK2) inhibitor, or Stattic, a selective signal transducer and activator of transcription 3 (STAT3) inhibitor. The cells were then stimulated with C6-ceramide. The levels of MMP-9 were determined by ELISA and real-time quantitative PCR (RT-qPCR). JAK2, phosphorylated JAK2 (p-JAK2), STAT3, and phosphorylated STAT3 (p-STAT3) expression was examined by Western blotting. BALB/c mice were pretreated with AG490 or Stattic before intratracheally instillated with C6-ceramide. Pathological changes in lung tissues were examined by Hematoxylin and Eosin staining, Periodic-acid Schiff staining, and Masson’s trichrome staining. MMP-9, JAK2, p-JAK2, STAT3, and p-STAT3 expression in the lung tissues was examined by Western blotting.ResultsThe expression of MMP-9, p-JAK2 and p-STAT3 in BEAS-2B cells was significantly increased after the treatment of C6-ceramide. Furthermore, the increased expression of MMP-9 induced by C6-ceramide was inhibited by AG490 and Stattic. Similar results were obtained in the lung tissues of C6-ceramide-exposed mice which were treated with AG490 or Stattic.ConclusionsCeramide could up-regulate MMP-9 expression through the activation of the JAK2/STAT3 pathway in airway epithelium. Targeted modulation of the ceramide signaling pathway may offer a potential therapeutic approach for inhibiting MMP-9 expression. This study points to a potentially novel approach to alleviating airway remodeling in inflammatory airway diseases.

Abstract 4907: A novel functional analysis of MET exon 14 skipping mutation in regulation of tumor cell invasion and metastasis

Abstract MET exon 14 skipping mutations (METΔ14Ex) represent a new class of molecular alterations that occur in approximately 3%-5% of non-small cell lung cancer (NSCLC) cases, and recently emerged as an actionable oncogenic driver with significant responsiveness to MET tyrosine kinase inhibitors, including crizotinib and capmatinib. METΔ14Ex mutations are generally was found to be mutually exclusive with other recognized oncogenic drivers and higher frequency has been noted in a unique, highly aggressive subtype of lung cancer called pulmonary sarcomatoid carcinoma. In the current study, we sought to perform an array of experiments to determine the intrinsic tumorigenic role of METΔ14Ex in NSCLC. Using multiple isogenic METΔ14Ex cell models established with CRISPR, we first determined that METΔ14Ex increased HGF-dependent kinase activity and enhanced HGF/MET downstream signaling through impairing receptor endocytic degradation. In subsequent functional analyses, we identified that METΔ14Ex significantly increases HGF-dependent cell scatter, migration and invasion capacity in vitro as well as metastasis in vivo compared to wild-type MET (MET WT). To explore the intrinsic mechanism in regulation of aforementioned phenotypes, an RNA sequencing study was conducted to evaluate the global changes of dynamic gene expression profile and key functional pathways. Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) revealed a large cluster of cell movement-related signaling pathways that were remarkably activated in HGF-treated METΔ14Ex models (vs MET WT) in a time-dependent manner. In addition, a number of upregulated DEGs (Differently Expressed Genes) were enriched in functional categories of cell cytoskeleton reorganization and cell-matrix adhesion degradation in METΔ14Ex models (vs MET WT). Based on the information of GSEA, we further found that the activity of the small GTPase Rac1, a crucial regulator of cytoskeleton dynamics, was significantly increased by HGF treatment in METΔ14Ex cells, whereas inhibition of the PI3K/Akt pathway repressed HGF/METΔ14Ex-mediated Rac1 activity and cell movement. Moreover, a group of Matrix metallopeptidases (MMPs) were highly upregulated in our models. Notably, among these MMPs, HGF-dependent upregulation of MMP1 expression (&amp;gt;100 fold in both mRNA and protein level) was very prominent in METΔ14Ex compared to MET WT cells, and silencing MMP1 using siRNA dramatically repressed HGF-triggered METΔ14Ex cell invasion. We finally found that the MET inhibitors, MGCD516 and capmatinib remarkably inhibited HGF/METΔ14Ex signaling and repressed cell invasion in vitro and metastasis in vivo. In conclusion, these data demonstrate that METΔ14Ex plays a critical role in enhancing cell movement and metastasis, at least partially via upregulation of the PI3K/Akt-Rac1 pathway and expression of MMPs, and blockade of HGF/METΔ14Ex signaling using MET inhibitors might be potent therapeutic options for METΔ14Ex-mutated NSCLC patients and recognition of the unique biology of METΔ14Ex-mediated oncogenesis might yield additional synergistic treatment strategies. Citation Format: Feng Wang, Yang Liu, Elaine Shum, Mariam Alexander, Deyou Zheng, Roman Perez-Soler, Haiying Cheng, Balazs Halmos. A novel functional analysis of MET exon 14 skipping mutation in regulation of tumor cell invasion and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4907.

Abstract 1521: New roles of ATP in metastasis: Extracellular and macropinocytosis internalized ATP work together to induces epithelial to mesenchymal transition and other early steps of metastasis

Abstract Metastasis is estimated to be responsible for more than 90% of all cancer-related deaths. Metastasis is initiated by induction of epithelial-mesenchymal transition (EMT) of cancer cells, but factors and mechanisms involved in EMT are far from being fully elucidated. Intratumoral extracellular ATP (eATP), at levels of 100-700 μM or 103 to 104 times higher than in normal tissues, has been known to induce epithelial-mesenchymal-transition (EMT) of cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis. Here we report that eATP, at the concentrations reported in tumors, potently induces expression of matrix metallopeptidases (MMPs), detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-β and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of the macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis and slows down tumor growth in nude mice. Collectively, these results show that eATP functions on these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings which shed light on eATP's initiator and effector roles in almost every step in early metastasis call for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identify eATP and macropinocytosis as novel targets for potentially slowing down EMT, preventing metastasis, and reducing metastasis-related death in cancer patients. Citation Format: Yanyang Cao, Xuan Wang, Maria Evers, Yunsheng Li, Haiyun Zhang, Xiaozhuo Chen. New roles of ATP in metastasis: Extracellular and macropinocytosis internalized ATP work together to induces epithelial to mesenchymal transition and other early steps of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1521.

Long non-coding RNA HCAR promotes endochondral bone repair by upregulating VEGF and MMP13 in hypertrophic chondrocyte through sponging miR-15b-5p

Endochondral bone formation is an important route for bone repair. Although emerging evidence has revealed the functions of long non-coding RNAs (lncRNAs) in bone and cartilage development, the effect of lncRNAs in endochondral bone repair is still largely unknown. Here, we identified a lncRNA, named Hypertrophic Chondrocyte Angiogenesis-related lncRNA (HCAR), and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13 (Mmp13) and vascular endothelial growth factor α (Vegfa) in hypertrophic chondrocytes. Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model. Mechanistically, we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization (FISH) assay, and it acted as a molecular sponge for miR-15b-5p. Further, in hypertrophic chondrocytes, lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression. Our results proved that lncRNA is deeply involved in endochondral bone repair, which will provide a new theoretical basis for future strategies for promoting fracture healing.

Upregulation of Tubulointerstitial nephritis antigen like 1 promotes gastric cancer growth and metastasis by regulating multiple matrix metallopeptidase expression.

Tubulointerstitial nephritis antigen-like 1 (TINAGL1), as a novel matricellular protein, has been demonstrated to participate in cancer progression, whereas the potential function of TINAGL1 in gastric cancer (GC) remains unknown. The expression pattern of TINAGL1 in GC was examined by immunohistochemistry, ELISA, real-time polymerase chain reaction, and Western blot. Correlation between TINAGL1 and matrix metalloproteinases (MMPs) was analyzed by the GEPIA website and Kaplan-Meier plots database. The lentivirus-based TINAGL1 knockdown, CCK-8, and transwell assays were used to test the function of TINAGL1 in vitro. The role of TINAGL1 was confirmed by subcutaneous xenograft, abdominal dissemination, and lung metastasis model. Microarray experiments, ELISA, real-time polymerase chain reaction, and Western blot were used to identify molecular mechanism. TINAGL1 was increased in GC tumor tissues and associated with poor patient survival. Moreover, TINAGL1 significantly promoted GC cell proliferation and migration in vitro as well as facilitated GC tumor growth and metastasis in vivo. TINAGL1 expression in GC cells was accompanied with increasing MMPs including MMP2, MMP9, MMP11, MMP14, and MMP16. GEPIA database revealed that these MMPs were correlated with TINAGL1 in GC tumors and that the most highly expressed MMP was MMP2. Mechanically, TINAGL1 regulated MMP2 through the JNK signaling pathway activation. Our data highlight that TINAGL1 promotes GC growth and metastasis and regulates MMP2 expression, indicating that TINAGL1 may serve as a therapeutic target for GC.

Polyphyllin II inhibits human bladder cancer migration and invasion by regulating EMT-associated factors and MMPs.

The epithelial-mesenchymal transition (EMT) serves vital roles in the angiogenesis, cell invasion and metastasis of various malignant tumors, including bladder cancer. Traditional Chinese medicinal herbs have been demonstrated to exhibit anticancer properties. The present study aimed to screen the sensitivity of bladder cancer to natural compounds by using six classic anti-inflammatory and detoxifying herbs, including the ethanol extract of Paris polyphylla (PPE), Scutellaria barbata, Pulsatillae decoction, Dahuang Huanglian Xiexin decoction, Bazhengsan and Hedyotis diffusa combined with S. barbata, were used to treat bladder cancer cells in vitro. Bladder cancer was more sensitive to PPE compared with the other tested herbs, and PPE significantly suppressed bladder cancer cell migration and invasion. Thus, the present study focused on PPE. Bladder cancer cells were treated with monomer components of PPE, including polyphyllin (PP) I, PPII, PPVI and PPVII. The results demonstrated that PPII treatment significantly inhibited cancer cell migration and invasion, increased the expression level of E-cadherin and decreased the levels of N-cadherin, snail family transcriptional repressor 2, twist family bHLH transcription factor 1, matrix metallopeptidase (MMP) 2 and MMP9 compared with those in the control group (untreated cells). These results suggested that PPII treatment may suppress bladder cancer cell migration and invasion by regulating the expression of EMT-associated genes and MMPs. Therefore, PPE and PPII may have antimetastatic effects and PPII may serve as a potential therapeutic option for inhibiting bladder cancer metastasis.

Apolipoprotein E isoforms differentially regulate matrix metallopeptidase 9 function in Alzheimer’s disease

Apolipoprotein E (APOE) has been shown to influence amyloid-β (Aβ) clearance from the brain in an isoform-specific manner. Our prior work showed that Aβ transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aβ elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aβ pathology in the AD brain.

Smooth Muscle Sirtuin 1 Blocks Thoracic Aortic Aneurysm/Dissection Development in Mice.

Advancing age is the major risk factor for thoracic aortic aneurysm/dissection (TAAD). However, the causative link between age-related molecules and TAAD remains elusive. Here, we investigated the role of Sirtuin 1 (SIRT1, also known as class III histone deacetylase), the best studied member of the longevity-related Sirtuin family, in TAAD development in vivo. We used male smooth muscle-specific SIRT1 transgenic (ST-Tg) mice, smooth muscle-specific SIRT1 knockout (ST-KO) mice, and their wild-type (WT) littermates on a C57BL/6J background to establish a TAAD model induced by oral administration of 3-aminopropionitrile fumarate (BAPN). We analyzed the incidence and fatality rates of TAAD in the groups. We examined matrix metallopeptidase 2 (MMP2) and MMP9 expression in aortas or cultured A7r5 cells via western blotting and real-time polymerase chain reaction (PCR). We performed chromatin immunoprecipitation (ChIP) to clarify the epigenetic mechanism of SIRT1-regulated MMP2 expression in vascular smooth muscle cells (VSMCs). BAPN treatment markedly increased the incidence of TAAD in WT mice but caused less disease in ST-Tg mice. Moreover, ST-KO mice had the highest BAPN-induced TAAD fatality rate of all the groups. Mechanistically, SIRT1 overexpression resulted in lower MMP2 and MMP9 expression after BAPN treatment in both mouse aortas and cultured A7r5 cells. The downregulation of BAPN-induced MMP2 expression by SIRT1 was mediated by deacetylation of histone H3 lysine 9 (H3K9) on the Mmp2 promoter in the A7r5 cells. Our findings suggest that SIRT1 expression in SMCs protects against TAAD and could be a novel therapeutic target for TAAD management.

BRD4 promotes metastatic potential in oral squamous cell carcinoma through the epigenetic regulation of the MMP2 gene

BackgroundOral squamous cell carcinoma (OSCC) has increased morbidity, and its high metastatic potential affects patient survival. Bromodomain containing 4 (BRD4) is a chromatin protein that associates with acetylated histone lysines and facilitates transcription. BRD4 has been implicated in cell proliferation, metastasis, and prognosis in several types of cancer. However, the role of BRD4 in OSCC remains to be elucidated.MethodsWe investigated the role of BRD4 and its potential utility as a therapeutic target in OSCC.ResultsJQ1, the BRD4 inhibitor, suppressed the cell proliferation, migration, and invasion in the OSCC cell lines and in vivo. JQ1 reduced the expression levels of 15 metastasis genes in OSCC, including matrix metallopeptidase 2 (MMP2). Our chromatin immunoprecipitation assay showed that JQ1 reduced the BRD4 binding to the histone H3 lysine 27 acetylation-enriched sites in the MMP2 locus. Analyses of biopsy specimens from OSCC patients revealed that the BRD4 and MMP2 expression levels were correlated in the cancerous regions, and both were highly expressed in lymph node metastasis cases, including delayed metastasis.ConclusionsBRD4 contributes to metastasis in OSCC, through the epigenetic regulation of the MMP2 gene, and thus BRD4 may represent a therapeutic target and a novel prediction indicator for metastasis.

LncRNA MIR4435-2HG inhibits the progression of osteoarthritis through miR-510-3p sponging.

Osteoarthritis (OA) is a disorder of diarthrodial joints that can have multiple causes. Long non-coding RNAs (lncRNAs) participate in multiple diseases, including OA. It has recently been reported that the lncRNA microRNA 4435-2HG (MIR4435-2HG) is downregulated in OA tissues; however, the biological role of MIR4435-2HG during OA progression remains unclear. In the present study, interleukin (IL)-1β was used to establish an in vitro model of OA. Protein expressions of matrix metallopeptidase (MMP) 1, MMP13, collagen II, interleukin (IL)-17A, p65, phosphorylated (p)-p65, IκB and p-IκB in CHON-001 cells were detected by western blotting. Gene expressions of IL-17A, MIR4435-2HG and miR-510-3p in tissues or CHON-001 cells were measured by reverse transcription-quantitative PCR and western blotting, respectively. Cell Counting Kit-8 assay and immunofluorescence staining were used to investigate cell proliferation, and cell apoptosis was detected by flow cytometry. The association between MIR4435-2HG, miR-510-3p and IL-17A was investigated using the dual luciferase report assay. MIR4435-2HG and miR-510-3p overexpression were transfected into CHON-001 cells. The results demonstrated that miR4435-2HG overexpression significantly increased proliferation and inhibited apoptosis of CHON-001 cells. In addition, miR-510-3p was identified as the downstream target of MIR4435-2HG, and miR-510-3p directly targeted IL-17A. The results from the present study suggested that MIR4435-2HG could mediate the progression of OA by inactivating the NF-κB signaling pathway. In addition, miR4435-2HG overexpression inhibited OA progression, suggesting that miR4435-2HG may be considered as a potential therapeutic target in OA.