MMP-9 Protein Levels Research Articles

Oxidative stress abrogates the degradation of KMT2D to promote degeneration in nucleus pulposus

Nucleus pulposus (NP) degeneration plays pivotal roles in intervertebral disc degeneration. The effect and mechanism of oxidative stress and epigenetics in NP degeneration is still unclear. We performed this study to evaluate the function of oxidative stress in NP and to explore the potential mechanism of ROS induced expression of matrix metalloproteinases (MMPs). We tested four methyltransferases, KMT2A, KMT2B, KMT2C and KMT2D in human NP samples, only KMT2D was significantly up-regulated in the severe degeneration samples. Knockdown of Kmt2d by siRNA significantly down-regulated the expression levels of catabolic enzymes including Mmp3, Mmp9 and Mmp13. Moreover, an interaction between KMT2D and ubiquitination was confirmed, and the application of H2O2 abrogated this process. Co-IP assay confirmed that H2O2 induced the phosphorylation of KMT2D to block the ubiquitination degradation, which was mainly mediated by phosphorylation of p38/MAPK. Further investigation suggested that ROS induced the alteration in levels of methylation is linked to H3K4me1 and H3K4me2, but not me3. However, usage of OICR-9429 (OICR) also suppressed the expression levels of Mmp3, Mmp9 and Mmp13. In an ex vivo model, application of OICR-9429 (OICR) also attenuated the degeneration of NP according to the H&E and Safranin-O/Fast Green staining assay, and the protein levels of MMP3, MMP9 and MMP13 were down-regulated, as well. In conclusion, we approved that oxidative stress induced ROS production promote the process of NP degeneration by enhancing KMT2D mediated transcriptional regulation of matrix degeneration related genes during NP degeneration.

A Comparison of Subconjunctival Wound Healing between Different Methods of Dissecting Subconjunctival Tissues

Objective: To compare different methods for dissecting subconjunctival tissues by developing subconjunctival wound healing models. Methods: New Zealand white rabbits were separated into 3 groups based on the method by which the rabbit subconjunctival wound healing model was generated: subconjunctival tissues were dissected episclerally (EPI) or subepithelially (SUB), with a corresponding blank control (CON). All the cases in the experimental groups were surgically prepared with conjunctival flaps, and they were sacrificed on the third postoperative day. At the surgical sites, the protein levels of hypoxia-inducible factor-1 (HIF-1)-α, vascular endothelial growth factor (VEGF)-A, and matrix metalloproteinase (MMP)-2 were detected by Western blot, morphological vascularity was measured by Adobe Photoshop, and subconjunctival fibrosis was assessed by histology. Results: Compared with the CON group, both the EPI and SUB groups showed significantly upregulated protein levels of HIF-1α, VEGF-A, and MMP-2. In addition, the protein levels of HIF-1α, VEGF-A, and MMP-2 were higher in the EPI group than in the SUB group. Morphological vascularity was significantly elevated in the EPI group compared with the SUB and CON groups. Collagen content was markedly increased in the EPI group compared with the SUB and CON groups. Conclusions: Dissecting subconjunctival tissues subepithelially inhibits subconjunctival fibrosis, which may be instructive in tenonectomy in filtration surgery.

Human mesenchymal stem cells respond differentially to platelet preparations and synthesize hyaluronic acid in nucleus pulposus extracellular matrix

BACKGROUND CONTEXTIn recent years, autologous platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) have been used as treatments for disc-related pain. A better understanding of the effects of leukocyte-rich (LR) versus leukocyte poor (LP-) PRP on bone marrow derived human mesenchymal stem/progenitor cells (hMSCs) is likely to improve future research studies, clinical practice and care for patients with chronic discogenic back pain. PURPOSEThe primary aim of this study is to determine the effects of LR-PRP and LP-PRP on the proliferation and migration of hMSCs in pig nucleus pulposus (NP) extracellular matrix (ECM). The secondary aim is to characterize hMSC-dependent expression of the matrix remodeling enzymes metalloproteinases MMP-2, MMP-3, MMP-9 and tissue inhibitor of metalloproteinases TIMP-2, and to determine whether transplanted hMSCs can synthesize hyaluronic acid (HA). STUDY DESIGNControlled laboratory study. METHODSBone marrow-derived culture expanded hMSCs were seeded onto pig NP and cultured with LR-PRP, LP-PRP or serum/platelet releasate (PR). The same conditions without hMSCs were used as controls. hMSC proliferation, migration and dispersion was assessed via fluorescent microscopy, while HA synthesis, MMP-2, MMP-3, MMP-9, and TIMP-2 protein levels were assessed via enzyme linked immunosorbent assay. All funding was provided by a 501c(3) research foundation and does not have any commercial or sponsorship interests. RESULTSLP-PRP and PR cultures resulted in higher hMSC proliferation, migration, dispersion, and MMP-2 expression. LP-PRP cultures resulted in the highest HA production. LR-PRP cultures resulted in lower hMSC proliferation, negligible migration and dispersion, increased MMP-9 expression and lower HA production. CONCLUSIONSHuman bone marrow-derived hMSCs seeded onto pig NP ECM are capable of synthesizing HA, indicating a transition towards a NP cell phenotype. This process was most enhanced by LP-PRP and marked by increased hMSC proliferation, MMP-2 production, HA synthesis and reduced MMP-9 levels. CLINICAL SIGNIFICANCELP-PRP and PR, with or without hMSCs, may provide better outcomes than LR-PRP in lab investigations and clinical trials for discogenic pain. Bone marrow-derived hMSCs may hold promise as a treatment for disc degeneration.

Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins α1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase β/ζ isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.

AB0070 LORECIVIVINT (SM04690), A POTENTIAL DISEASE-MODIFYING TREATMENT FOR KNEE OSTEOARTHRITIS, DEMONSTRATED CARTILAGE-PROTECTIVE EFFECTS ON HUMAN OSTEOARTHRITIC EXPLANTS

Background:Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteocyte differentiation, cartilage thinning, and inflammation. Lorecivivint (LOR; SM04690), a novel, small-molecule CLK/DYRK1A inhibitor that modulates the Wnt pathway, demonstrated disease-modifying potential for knee OA in preclinical studies.1However, the specific mechanisms by which LOR protects cartilage in knee OA are unclear.Objectives:To evaluate the cartilage-protective effects of LOR on human OA explants from total knee replacement (TKR) donors.Methods:Knee joint tissue from 22 TKR donors was obtained. IRB approval was obtained from Scripps Health. Cartilage was scored using the Outerbridge classification system based on gross appearance (grade 1=least-damaged tissue, grade 4=most-damaged tissue). Cartilage explants (4 mm in diameter) with Outerbridge grades 2–3 were harvested and cultured for 48 hours to reach metabolic stability. They were then treated with LOR (10 nM, 30 nM) or DMSO and stimulated with either IL-1β (10 ng/ml) or TNF-α (20 ng/ml)+oncostatin M (OM) (10 ng/ml) or left unstimulated. After 72 hours, supernatants and explants were collected. Gene expression of matrix metalloproteinases (MMPs) 1, 3, and 13 was measured by qPCR and protein levels of MMP-1, MMP-3, MMP-13, and thrombospondin-motif-containing disintegrins/metalloproteinases ADAMTS-4 and ADAMTS-5 were measured in supernatants by ELISA. Glycosaminoglycan (GAG) and nitric oxide (NO) levels were measured in supernatants using the dimethylmethylene blue assay and Griess assay, respectively. One-way ANOVA was used for multiple group comparisons.Results:Treatment with IL-1β or TNF-α+OM led to statistically significant increases in gene expression ofMMP1,MMP3, andMMP13and increased secretion of GAG, MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and NO in supernatants compared to unstimulated control. Treatment with LOR decreased both IL-1β-stimulated and TNF-α+OM-stimulated gene expression ofMMP1,MMP3, andMMP13and secretion of GAG, MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and NO in supernatants compared to treatment with DMSO.Conclusion:LOR demonstrated potent inhibition of cartilage catabolism enzyme production in human OA explants compared to controls. These cartilage-protective effects support the development of LOR as a potential disease-modifying treatment for knee OA. Human trials are ongoing.

Exosomal Transfer of lncRNA H19 Promotes Erlotinib Resistance in Non-Small Cell Lung Cancer via miR-615-3p/ATG7 Axis.

BackgroundDrug resistance restrains the effect of drug therapy in non-small cell lung cancer (NSCLC). However, the mechanism of the acquisition of drug resistance remains largely unknown. This study aims to investigate the effect of exosomal lncRNA H19 on erlotinib resistance in NSCLC and the underlying mechanism.MethodsHCC827 and A549 cells were continuously grafted into erlotinib-containing culture medium to establish erlotinib-resistant cell lines. The expression of H19 and miR-615-3p was detected by qRT-PCR. The protein levels of MMP2, MMP9, CD9, CD63 and ATG7 were measured by Western blot. Cell viability and proliferation were determined by Cell Counting Kit-8 (CCK-8) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, respectively. Migration and invasion were assessed by transwell assay. Xenograft tumor models were used to investigate the effect of H19 on erlotinib resistance in vivo. Online software and dual-luciferase reporter assay were used to predicate the downstream targets and confirm the targeted relationships.ResultsH19 was upregulated in erlotinib-resistant cells, and knockdown of H19 inhibited cell proliferation, migration and invasion in erlotinib-resistant cells. Extracellular H19 can be packaged into exosomes. Exosomes containing H19 induced erlotinib resistance of sensitive cells, while knockdown of H19 abolished this effect. miR-615-3p was a target of H19 and can bind to ATG7. Exosomal H19 affected erlotinib resistance of erlotinib-resistant NSCLC cells via targeting miR-615-3p to regulate ATG7 expression. In addition, the serum exosomal H19 was upregulated in patients with erlotinib resistance. Furthermore, downregulated H19 decreased the resistance of tumor cells to erlotinib in vivo.ConclusionOur study demonstrated that exosomal H19 facilitated erlotinib resistance in NSCLC via miR-615-3p/ATG7 axis, which might provide a potential target for the diagnosis and treatment of NSCLC.

Enhancer of zeste homolog 2 (EZH2) gene inhibition via 3-Deazaneplanocin A (DZNep) in human liver cells and it is relation with fibrosis

Abstract Objectives Hepatic fibrosis is a complex and dynamic process, such as “wound healing”. The effect of 3-deazaneplanocin A (DZNep) via enhancer of zeste homolog 2 (EZH2) inhibition on transforming growth factor, matrix metalloproteinases 2-9 (MMP2, MMP9) and matrix metalloproteinases inhibitor 3 (TIMP3), alpha-smooth muscle actin (α-SMA), collagen 1A1(COL1A1), and collagen 3A1 (COL3A1) genes/proteins in human hepatic stellate cell line were examined. Inhibition effect of EZH2 on colony formation and migration were investigated. Methods 5 μM DZNep was treated to LX2 cells for 3 days. Real time PCR and Western blot method were used for analyses. Results DZNep was shown to reduce colony formation and migration. It increased the α-SMA only in gene expression level but decreased at the protein levels. It down-regulated both gene expression and protein levels of EZH2, while up-regulated gene expression and protein levels of TGF-β. DZNep induced gene expression and protein levels of MMP2 and TIMP3. However, DZNep induced COL1A1 and COL3A1 gene expression levels but reduced protein levels only in COL3A1 but not COL1A1. Conclusion DZNep has a positive anti-fibrotic effect by reducing α-SMA and COL3A1 protein levels in the LX2 cell line, however, we have found that DZNep may also has fibrotic effect because it increases TGF-β both expression and protein levels.

Elevated MMP9 expression in breast cancer is a predictor of shorter patient survival

PurposeMMP9 is a matricellular protein associated with extracellular matrix (ECM) remodelling, that promotes tumour progression, and modulates the activity of cell adhesion molecules and cytokines. This study aims to assess the prognostic value of MMP9 and its association with cytoskeletal modulators in early-stage invasive breast cancer (BC).MethodsMMP9 expression was evaluated by immunohistochemistry using a well-characterised series of primary BC patients with long-term clinical follow-up. Association with clinicopathological factors, patient outcome and ECM remodelling BC-biomarkers were investigated. METABRIC dataset, BC-GenExMiner v4.0 and TCGA were used for the external validation of MMP9 expression. GSEA gene enrichment analyses were used to evaluate MMP9 associated pathways.ResultsMMP9 immunopositivity was observed in the stroma and cytoplasm of BC cells. Elevated MMP9 protein levels were associated with high tumour grade, high Nottingham Prognostic Index, and hormonal receptor negativity. Elevated MMP9 protein expression correlated significantly with cytokeratin 17 (Ck17), Epidermal Growth Factor Receptor (EGFR), proliferation (Ki67) biomarkers, cell surface adhesion receptor (CD44) and cell division control protein 42 (CDC42). Cytoplasmic MMP9 expression was an independent prognostic factor associated with shorter BC-specific survival. In the external validation cohorts, MMP9 expression was also associated with poor patients’ outcome. Transcriptomic analysis confirmed a positive association between MMP9 and ECM remodelling biomarkers. GSEA analysis supports MMP9 association with ECM and cytoskeletal pathways.ConclusionThis study provides evidence for the prognostic value of MMP9 in BC. Further functional studies to decipher the role of MMP9 and its association with cytoskeletal modulators in BC progression are warranted.

Rubimaillin suppresses proliferation, migration and invasion of prostate cancer cells via the Notch-1/MMP signaling pathway

This study was aimed to explore the effect of Rubimaillin on the survival, migration, and invasion of prostate cancer cell lines DU145 and PC3, and its mechanism. CCK-8 method was used to detect the effects of different concentrations of rubs (0, 5, 10, 20, 40 and 80 μM) on the activity of DU145 cells and PC3 cells. Transwell cell lab test was used to detect the migration and invasion of cells. Western blot was used to detect Notch-1, MMP-2, MMP-9 and Hes-1 protein levels. The CCK-8 assay showed that Rub inhibited the activity of Du145 and PC3 cells in a concentration dependent manner. When the concentration reached 40 μM, the inhibition reached the maximum. After Rub intervention, the migration and invasion ability of Du145 and PC3 cells decreased significantly, while the expression levels of Notch-1, MMP-2, MMP-9 and Hes-1 protein decreased significantly. Rub can inhibit the growth, migration and invasion of prostate cancer cell line DU145 and PC3. The mechanism may be related to the inhibition of Notch-1, MMP-2, MMP-9 and Hes-1 by Rub.

NF-κB inhibitor, NEMO-binding domain peptide attenuates intervertebral disc degeneration

BACKGROUND CONTEXTNonphysiological mechanical loading and inflammation are both critically involved in intervertebral disc (IVD) degeneration, which is characterized by an increase in cytokines and matrix metalloproteases (MMPs) in the nucleus pulposus (NP). This process is known to be mediated by the NF-κB pathway. CLINICAL SIGNIFICANCECurrent clinical treatments for IVD degeneration focus on the alleviation of symptoms rather than targeting the underlying mechanism. Injection of an NF-κB inhibitor may attenuate the progression of IVD degeneration. PURPOSETo investigate the ability of the NF-κB inhibitor, NEMO binding domain peptide (NBD), to alter IVD degeneration processes by reducing IL-1β- and mechanically-induced cytokine and MMP levels in human nucleus pulposus cells in vitro, and by attenuating IVD degeneration in an in vivo rat model for disc degeneration. STUDY DESIGNExperimental in vitro and animal model. PATIENT SAMPLEDiscarded specimens of lumbar disc from 21 patients, and 12 Sprague Dawley rats. OUTCOME MEASURESGene and protein expression, cell viability, µMRI and histology. METHODSIL-1β-prestimulated human nucleus pulposus cells embedded into fibrin constructs were loaded in the Flexcell FX-5000 compression system at 5 kPa and 1 Hz for 48 hours in the presence and absence of NBD. Unloaded hNPC/fibrin constructs served as controls. Cell viability in loaded and unloaded constructs was quantified, and gene and protein expression levels determined. For in vivo testing, a rat needle disc puncture model was employed. Experimental groups included injured discs with and without NBD injection and uninjured controls. Levels of disc degeneration were determined via µMRI, qPCR and histology. Funding sources include $48,874 NASS Young Investigator Research Grant and $119,174 NIH 5K01AR071512-02. There were no applicable financial relationships or conflicts of interest. RESULTSMechanical compression of hNPC/fibrin constructs resulted in upregulation of MMP-3 and IL-8. Supplementation of media with 10 μM NBD during loading increased cell viability, and decreased MMP-3 gene and protein levels. IVD injury in rat resulted in an increase in MMP-3, IL-1β and IL-6 gene expression. Injections of 250 µg of NBD during disc injury resulted in decreased IL-6 gene expression. µMRI analysis demonstrated a reduction of disc hydration in response to disc needle injury, which was attenuated in NBD-treated IVDs. Histological evaluation showed NP and AF lesion in injured discs, which was attenuated by NBD injection. CONCLUSIONSThe results of this study show NBD peptide's capacity to reduce IL-1β- and loading-induced MMP-3 levels in hNPC/fibrin constructs while increasing the cells’ viability, and to attenuate IVD degeneration in rat, involving downregulation of IL-6. Therefore, NBD may be a potential therapeutic agent to treat IVD degeneration.

Blocking Wnt/β-Catenin Signaling Alleviates Osteoarthritis in Rats

Osteoarthritis is a chronic multiple degenerative bone and joint disease. The most common manifestations are damaged articular cartilage, thickening and sclerosis of subchondral bone, and osteophytes formation at the joint edge. This study will block the Wnt/β -catenin signaling pathway to observe its role on a rat model of osteoarthritis. In this experiment, rat osteoarthritis model was established by intra-articular administration of iodoacetic acid. The LF-3 treatment was started on the second day after modeling. Two weeks later, rat knee articular cartilage was collected. The morphology of cartilage was observed by HE, Safranin O and toluidine blue staining. The expression levels of TIMP-3, MMP-9 protein and mRNA were detected by western blot and real-time quantitative PCR. After blocking the Wnt/ β-catenin signaling pathway, TIMP-3 expression level was increased, MMP-9 expression was decreased, and cartilage damage was ameliorated. Blocking the Wnt/ β-catenin signal can reduce the degradation of matrix in cartilage tissue, thereby playing a protective role and helping the recovery of osteoarthritic rats.

Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing.

Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100nm and a positive zeta potential about 40mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.

Suppression of experimental atrial fibrillation in a canine model of rapid atrial pacing by the phosphodiesterase 3 inhibitor cilostazol

ObjectiveAtrial fibrillation (AF) represents the most common arrhythmia encountered in cardiology department. The purpose of this study was designed to investigate whether cilostazol, an oral phosphodiesterase 3 inhibitor (PDE3) could have protective effects on atrial remodeling in a canine model of AF and explore the potential molecular mechanisms. MethodsDogs were randomly assigned to Sham, Paced, Paced + cilostazol group, 7 dogs in each group. In Sham group, pacemaker was instrumented but without pacing. Rapid atrial pacing (RAP) at 600 or 500 bpm/min was maintained in Paced group and Paced + cilo group for 2 h or 2 weeks in acute or chronic experiment, respectively. The Paced + cilo group of dogs were pretreated with cilostazol orally (10 mg·kg−1·d−1, cilo) for 1 h or 2 days prior RAP induction and served as treatment group. Atrial effective refractory periods (AERP) at different basic cycle lengths (BCLs), inducibility, and duration time of AF were measured after pacing for 2 h. The blood sample, echocardiography, histopathology, inflammation and oxidative stress makers, protein and mRNA expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected after 2 weeks pacing in each group. ResultsSignificant changes in electrophysiological parameters were observed in the acute RAP canine model, the AERPs shortened with increased inducibility and duration of AF, which was attenuated by cilostazol (P < 0.05). The serum inflammation makers as interleukin-8 (IL-8) and toll like receptor 4 (TLR 4) levels and oxidative stress indicators like xanthine oxidative (XO) and reactive oxygen species (ROS) in the Paced group was significantly higher than that in Sham group (P < 0.01), and was significantly reduced by cilostazol treatment (P < 0.01). The level of mean platelet volume (MPV) which is one of the platelet indices was significantly elevated in Paced group (P < 0.01). While after cilostazol treated for 2 weeks, the level of MPV was obviously decreased than Paced group (P < 0.01). Pathology and echocardiography studies showed that cilostazol can also prevent RAP induced cardiac fibrosis and structural remodeling. The MPV level has close correlations with IL-8, TLR4, XO and ROS (all P < 0.01). MMP-2 and MMP-9 expression were significantly increased in Paced group (all P < 0.01), which can be attenuated by cilostazol. ConclusionsCilostazol may have protective effects on RAP-induced atrial remodeling by anti-inflammatory, anti-oxidative stress action and regulate the extracellular collagen matrix in a canine model. Moreover, MPV level is associated with inflammation and oxidative stress response of RAP, which might be an important predictors of new-onset and recurrent AF.

MiR124-3p/FGFR2 axis inhibits human keratinocyte proliferation and migration and improve the inflammatory microenvironment in psoriasis

Keratinocyte hyperproliferation has been regarded as a central event in psoriasis pathogenesis. Investigating the mechanisms of keratinocyte hyperproliferation might provide novel strategies for psoriasis treatment. we demonstrated that fibroblast growth factor receptor 2 (FGFR2) expression was abnormally upregulated within psoriatic lesion tissues and HaCaT cells under rIL-22 stimulation. FGFR2 silence within HaCaT cells under rIL-22 stimulation significantly inhibited the capacity of cells to proliferate and to migrate, reduced IL-17A and TNFα mRNA expression, and decreased the protein levels of FGFR2, keratin 6, keratin 16, MMP1, MMP9, p-PI3K, p-AKT and p-ERK. In contrast to FGFR2, the expression of miR-124-3p showed to be remarkably downregulated within psoriasis lesion tissue samples and rIL-22-stimulated HaCaT cells. miR-124-3p inhibited the expression of FGFR2 via direct binding to its 3'UTR. Within HaCaT cells under rIL-22 stimulation, the overexpression of miR-124-3p also suppressed the capacity of cells to proliferate and to migrate, reduced IL-17A and TNFα mRNA expression, and decreased the protein levels of FGFR2, keratin 6, keratin 16, MMP1, MMP9 and p-PI3K, p-AKT and p-ERK. More importantly, when co-transfected to HaCaT cells, FGFR2-overexpressing vector significantly attenuated the effects of miR-124-3p mimics on HaCaT cells. In conclusion, we demonstrated an miR124-3p/FGFR2 axis that might inhibit human keratinocyte proliferation, migration, and improve the inflammatory microenvironment in psoriasis. miR124-3p/FGFR2 axis could be an underlying target for psoriasis therapy, which requires further in vivo and clinical investigation.

Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line.

Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM.

Features of Stable Plaque Phenotype Are Increased by Macrophage Specific Insulin‐Like Growth Factor 1

Insulin‐like growth factor I (IGF‐1) reduces atherosclerosis in Apoe‐null mice and promotes stable plaque phenotype. We reported that deficiency for macrophage (MF)‐specific IGF‐1 receptor upregulated matrix metalloproteinases (MMPs) and promoted atherogenesis. To test whether MF‐specific IGF‐1 will suppress MMPs, decrease atherosclerosis, and change plaque stability, we generated Apoe‐null mice with IGF‐1 overexpression driven by MF‐specific promoter (scavenger receptor A) (SRA12 and SRA17 mice). IGF‐1 level was increased in peritoneal MF isolated from SRA12 (21±4% increase) and from SRA17 mice (52±9% increase) compared to control Apoe‐null mice (both P&lt;0.05). SRA12 and SRA17 mice were fed with a high fat diet for 12 weeks. MF‐specific IGF‐1 overexpression significantly decreased atherosclerotic burden (SRA12, 23±3% decrease, n=20, and SRA17, 28±6% decrease, n=16, vs. control, n=29, en face analysis) and increased plaque collagen levels (11.1±6%, SRA12 and 66.5±11%, n=11, SRA17, Trichrome). Atherosclerotic plaques in both SRA12 and SRA17 mice had reduced necrotic cores suggesting elevated plaque stability. Increased levels of plaque collagen in SRA17 mice correlated with a significant decrease of MMP1, MMP2, MMP8, MMP12, MMP13, and MMP14 protein levels in peritoneal MF (immunoblotting, 30–72% decrease vs. control). To assess monocyte recruitment into the plaque, macrophage‐cleavable fluorescent red beads were injected (IV). Plaques in both SRA12 and SRA17 mice have reduced number of red beads compared to Apoe‐null mice (77±2% decrease, p=0.027, 74±3% decrease, p=0.0592, respectively) suggesting decreased monocyte recruitment. Cutlured human THP‐1 MFs were incubated with IGF‐1 (0, 20, 100ng/mL) for 48 hrs. IGF‐1 (20ng/ml) decreased expression of MMP8 (34±13% decrease, P&lt;0.05) and MMP14 protein (35±17% decrease, P&lt;0.05) (ELISA). To demonstrate that downregulation of MMPs was mediated via IGF1R‐dependent mechanism, we used Picropodophyllin (PPP), a specific IGF1R inhibitor. MF pre‐treatment with PPP (1ug/ml, 2hrs) abolished IGF‐1‐induced MMP8 downregulation in MF. In summary, we show that IGF‐1 reduced MMPs in vitro and MF IGF‐1 decreased MMPs, upregulated collagen and suppressed atherosclerotic burden in vivo. We identified the novel MMP‐dependent mechanism potentially mediating MF IGF‐1 atheroprotective effect by increasing features of stable plaque phenotype.Support or Funding InformationNIH/NHLBI 2R01HL070241‐13A1

Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury.

ObjectiveThis study aimed to examine the changes and significance of matrix metalloproteinase 9 (MMP9), MMP2, tissue inhibitor of metalloproteinase 1 (TIMP1), and TIMP2 in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsWistar rats were randomly divided into a control group (injected with saline) and an ALI group (injected with LPS), then subdivided into four time points (2, 6, 12, and 24 hours). Serum tumor necrosis factor alpha and interleukin-6 levels were detected by ELISA to investigate the inflammatory reaction after LPS injection. The degree of ALI was determined by hematoxylin–eosin staining of lung tissue, the lung wet/dry weight ratio, and pulmonary permeability index. Changes in lung MMP and TIMP protein and mRNA levels were detected by western blotting and quantitative real-time polymerase chain reaction.ResultsChanges in the ratios of MMP9/TIMP1 and MMP2/TIMP2 were consistent with and strongly positively associated with the lung wet/dry weight ratio, the pulmonary permeability index, and serum tumor necrosis factor alpha and interleukin-6 levels in the ALI group.ConclusionALI induced by LPS may be related to upregulation of MMP9/TIMP1 and MMP2/TIMP2 ratios.

Ivermectin suppresses tumour growth and metastasis through degradation of PAK1 in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC), the most common form of oesophageal malignancies in the Asia‐Pacific region, remains a major clinical challenge. In this study, we found that ivermectin, an effective antiparasitic drug that has been approved for patients to orally treat onchocerciasis for over 30 years, displayed potent antitumour activity against ESCC cells in vitro and in nude mice. We demonstrated that ivermectin significantly inhibited cell viability and colony formation, and induced apoptosis through a mitochondrial‐dependent manner in ESCC cells. Ivermectin also abrogated ESCC cell migration, invasion, as well as the protein levels of MMP‐2 and MMP‐9. Mechanistically, ivermectin strongly inhibited the expression of PAK1; by further gain‐ and loss‐of‐function experiments, we confirmed that PAK1 played a crucial role in ivermectin‐mediated inhibitory effects on ESCC cells. In addition, the data indicated that ivermectin promoted PAK1 degradation through the proteasome‐dependent pathway. Additionally, ivermectin synergized with chemotherapeutic drugs including cisplatin and 5‐fluorouracil to induce apoptosis of ESCC cells. Interestingly, the in vivo experiments also confirmed that ivermectin effectively suppressed tumour growth and lung metastasis of ESCC. Collectively, these results indicate that ivermectin exerts a potent antitumour activity against ESCC and is a promising therapeutic candidate drug for ESCC patients, even those carrying metastasis.

Galectin-1 Overexpression Activates the FAK/PI3K/AKT/mTOR Pathway and Is Correlated with Upper Urinary Urothelial Carcinoma Progression and Survival.

Galectin-1 (GAL1) is a β-galactoside-binding protein involved in multiple aspects of tumorigenesis. However, the biological role of GAL1 in upper tract urothelial carcinoma (UTUC) has not been entirely understood. Herein, we investigated the oncological effects of GAL1 expression in tumor specimens and identified related gene alterations through molecular analysis of GAL1. Clinical parameter data and tumor specimens were collected from 86 patients with pT3N0M0 UTUC who had undergone radical nephroureterectomy. We analyzed the difference in survival by using Kaplan–Meier analyses and Cox proportional regression models and in GAL1 expression by using immunohistochemical (IHC) methods. Public genomic data from the Cancer Genome Atlas (TCGA) and GSE32894 data sets were analyzed for comparison. Using four urothelial carcinoma (UC) cell lines (BFTC-909, T24, RT4, and J82) as in vitro models, we evaluated the functions of GAL1 in UC cell growth, invasiveness, and migration and its role in downstream signaling pathways. The study population was classified into two groups, GAL1-high (n = 35) and GAL1-low (GAL1 n = 51), according to IHC interpretation. Univariate analysis revealed that high GAL1 expression was significantly associated with poor recurrence-free survival (RFS; p = 0.028) and low cancer-specific survival (CSS; p = 0.025). Multivariate analysis revealed that GAL1-high was an independent predictive factor for RFS (hazard ratio (HR) 2.43; 95% confidence interval (CI) 1.17–5.05, p = 0.018) and CSS (HR 4.04; 95% CI 1.25–13.03, p = 0.019). In vitro studies revealed that GAL1 knockdown significantly reduced migration and invasiveness in UTUC (BFTC-909) and bladder cancer cells (T24). GAL1 knockdown significantly reduced protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9, which increased tissue inhibitor of metalloproteinase-1 (TIMP-1) and promoted epithelial–mesenchymal transition (EMT). Through gene expression microarray analysis of GAL1 vector and GAL1-KD cells, we identified multiple significant signaling pathways including p53, Forkhead box O (FOXO), and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). We validated microarray results through immunoblotting, thus proving that downregulation of GAL1 reduced focal adhesion kinase (FAK), p-PI3K, p-AKT, and p-mTOR expression. We concluded that GAL1 expression was highly related to oncological survival in patients with locally advanced UTUC. GAL1 promoted UC invasion and metastasis by activating the FAK/PI3K/AKT/mTOR pathway.

3,5-Dicaffeoyl-epi-quinic acid inhibits the PMA-stimulated activation and expression of MMP-9 but not MMP-2 via downregulation of MAPK pathway.

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, are very important gelatinases that are overexpressed during tumor metastasis. Up to date, several MMP inhibitors have been developed from natural sources as well as organic synthesis. In the present study, the MMP-2 and MMP-9 inhibitory effects of 3,5-dicaffeoyl-epi-quinic acid (DCEQA), a caffeoylquinic acid derivative isolated from Atriplex gmelinii, were investigated in phorbol 12-myristate 13-acetate (PMA)-treated human HT1080 fibrosarcoma cells. Gelatin zymography and immunoblotting showed that DCEQA significantly inhibited the PMA-induced activation and expression of MMP-9 but was not able to show any effect against MMP-2. DCEQA treatment was also shown to upregulate the protein expression of tissue inhibitor of MMP-1 along with decreased MMP-9 protein levels. Moreover, the effect of DCEQA on phosphorylation of mitogen activated protein kinases (MAPKs), analyzed by immunoblotting, indicated the DCEQA inhibited the MMP-9 by downregulation of MAPK pathway. Collectively, current results suggested that DCEQA is a potent MMP-9 inhibitor and can be utilized as lead compound for treatment of pathological complications involving enhanced MMP activity such as cancer metastasis.