Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-kB and matrix metalloproteinase-2/-9-signaling pathways.

Metastasis plays an important role in mortality of cancer patients. Migration and invasion are the major characteristics of tumor metastasis. The induction of matrix metalloproteinases (MMPs) such as MMP-2 and -9 are particularly important for the invasiveness of various cancer cells. Bufalin, a class of toxic steroids, was purified from the skin glands of Bufo gargarizans or Bufo melanostictus; it is known to inhibit proliferation of human cancer cells. In this study, we investigated the antiinvasive mechanisms of bufalin in the human hepatocellular cancer cell line SK-Hep1. Bufalin significantly reduced serum-induced cell invasion and migration. Furthermore, bufalin markedly inhibited MMP-2 and -9 activity, mRNA expression and protein levels in SK-Hep1 cells. Bufalin attenuated phosphoinisitide-3-kinase (PI3K) and phosphorylation of AKT which was associated with reduced levels of nuclear factor kappa B (NF-κB). Bufalin also suppressed protein levels of FAK and Rho A, VEGF, MEKK3, MKK7, and uPA and it diminished NF-κB translocation. Based on these observations, we propose that bufalin is acts as an antiinvasive agent by inhibiting MMP-2 and -9 and involving PI3K/AKT and NF-κB pathways. Bufalin is a potential therapeutic agent that may have efficacy in preventing the invasion and metastasis of malignant liver tumors.

Sinulariolide Suppresses Human Hepatocellular Carcinoma Cell Migration and Invasion by Inhibiting Matrix Metalloproteinase-2/-9 through MAPKs and PI3K/Akt Signaling Pathways.

Sinulariolide is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigate the migration and invasion effects of sinulariolide in hepatocellular carcinoma cell HA22T. Sinulariolide inhibited the migration and invasion effects of hepatocellular carcinoma cells in a concentration-dependent manner. The results of zymography assay showed that sinulariolide suppressed the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Moreover, protein levels of MMP-2, MMP-9, and urokinase-type plasminogen activator (uPA) were reduced by sinulariolide in a concentration-dependent manner. Sinulariolide also exerted an inhibitory effect on phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, Focal adhesion kinase (FAK), growth factor receptor-bound protein 2 (GRB2). Taken together, these results demonstrated that sinulariolide could inhibit hepatocellular carcinoma cell migration and invasion and alter HA22T cell metastasis by reduction of MMP-2, MMP-9, and uPA expression through the suppression of MAPKs, PI3K/Akt, and the FAK/GRB2 signaling pathway. These findings suggest that sinulariolide merits further evaluation as a chemotherapeutic agent for human hepatocellular carcinoma.

Association of Serum Dystroglycan, MMP-2/9 and AQP-4 with Haematoma Expansion in Patients with Intracerebral Haemorrhage.

OBJECTIVE: The purpose of this study was to explore association of serum dystroglycan (DG), matrix metalloproteinase-2/matrix metalloproteinase-9 (MMP-2/9), and aquaporin-4 (AQP-4) expression and haematoma expansion in patients with intracerebral haemorrhage (ICH), which are proteins involved in maintaining the integrity of the blood-brain barrier. METHODS: We included patients older than 18 years old with ICH who had undergone baseline CT within 6 hours after intracerebral haemorrhage symptom onset in our hospital between April 2018 and December 2018. Two readers independently assessed haematoma volume and other imaging information upon admission and again within 24 hours. All patients underwent 5 mL of venous blood collection 6 and 24 hours after admission. Serum expression levels of dystroglycan, matrix metalloproteinase-2/matrix metalloproteinase-9 and aquaporin-4 were determined by quantitative enzyme-linked immunosorbent assay (ELISA). Repeated analysis of variance was used to determine whether expression of the four proteins in patients with cerebral haemorrhage changed within 24 hours and whether there were differences between the haematoma enlargement and non-haematoma enlargement groups over time. Univariate and multivariate logistic regression analyses were used to compare the correlation among expression of the four proteins, clinical characteristics of patients and haematoma enlargement. RESULTS: Expression levels of serum matrix metalloproteinase-2/matrix metalloproteinases-9 and aquaporin-4 gradually increased within 24 hours in patients with cerebral haemorrhage (P<0.001), while expression levels of dystroglycan gradually decreased (P<0.01). Expression of serum matrix metalloproteinases-9 6 hours after onset was independently correlated with the expansion of cerebral haemorrhage. The ROC curve (AUC=0.778, 95% Cl: 0.661-0.894, P<0.001) exhibited high sensitivity (0.900) and low specificity (0.642). CONCLUSION: These data support that expression of MMP-9 in peripheral blood is independently correlated with the enlargement of haematoma in patients with intracerebral haemorrhage 6 hours after onset and can be used as an independent predictor of haematoma enlargement in patients with intracerebral haemorrhage. However, although the expression of MMP-2, AQP-4 and DG exhibited some changes within 6 and 24 hours after onset, they were not independently correlated with early haematoma enlargement in patients with intracerebral haemorrhage. Further multi-time point exploration and expansion of the sample size is necessary in future studies.

Water treadmill training attenuates blood-spinal cord barrier disruption in rats by promoting angiogenesis and inhibiting matrix metalloproteinase-2/9 expression following spinal cord injury.

BACKGROUND: The permeability of the blood-spinal cord barrier (BSCB) is mainly determined by junction complexes between adjacent endothelial cells (ECs), including tight junctions (TJs) and adherens junctions (AJs), which can be severely damaged after spinal cord injury (SCI). Exercise training is a recognized method for the treatment of SCI. The destruction of the BSCB mediated by matrix metalloproteinases (MMPs) leads to inflammation, neurotoxin production, and neuronal apoptosis. The failure of new blood vessels to effectively regenerate is also an important cause of delayed recovery after SCI. For the first time, we introduced water treadmill training (TT) to help SCI rats successfully exercise and measured the effects of TT in promoting recovery after SCI and the possible mechanisms involved. METHODS: Sprague-Dawley (200-250 g) rats were randomly divided into the following three groups: sham operated, SCI, and SCI + TT. Animals were sacrificed at 7 or 14 days post-surgery. The degree of neurological deficit, tissue morphology and BSCB permeability were assessed by the Basso-Beattie-Bresnahan (BBB) motor function scale and appropriate staining protocols, and apoptosis, protein expression and vascular EC ultrastructure were assessed by TUNEL staining, Western blotting, immunofluorescence and transmission electron microscopy (TEM). RESULTS: Our experiments showed that TT reduced permeability of the BSCB and decreased structural tissue damage. TT significantly improved functional recovery when compared with that in the SCI group; TJ and AJ proteins expression increased significantly after TT, and training reduced apoptosis induced by SCI. TT could promote angiogenesis, and MMP-2 and MMP-9 expression was significantly inhibited by TT. CONCLUSIONS: The results of this study indicate that TT promotes functional recovery for the following reasons: TT (1) protects residual BSCB structure from further damage, (2) promotes vascular regeneration, and (3) inhibits MMP-2/9 expression to mitigate BSCB damage.

CircLMNB1 promotes colorectal cancer by regulating cell proliferation, apoptosis and epithelial-mesenchymal transition.

OBJECTIVE: The aberrant expression of circular RNAs (circRNAs) is a frequent occurrence in various cancers. However, the functions and roles played by most circRNAs in colorectal cancer (CRC) remain largely unknown. MATERIALS AND METHODS: Levels of circLMNB1 expression were evaluated by qRT-PCR and FISH assays. The influence of circLMNB1 knockdown on LoVo and HCT116 cell proliferation, cycling, apoptosis, migration, and invasion were assessed by the CCK-8, assay, Edu assay, flow cytometry, Hoechst staining, and the Transwell assay, respectively. The relative levels of EMT- and apoptosis-related proteins were determined by Western blotting. RESULTS: CircLMNB1 expression was significantly upregulated in CRC tissues and cells. Knockdown of circLMNB1 by siRNA in LoVo cells suppressed cell proliferation, migration and invasion, and facilitated cell cycle arrest and apoptosis In addition, we proved that knockdown of circLMNB1 upregulated E-cadherin, Bax and caspase-3 expression, and downregulated MMP2, MMP-9, and N-cadherin expression in LoVo cells. Further results showed that overexpression of circLMNB1 enhanced the malignant characteristics of HCT116 cells. CONCLUSION: Our findings revealed that blocking of circLMNB1 could inhibit CRC development, and help to explain the underlying mechanism by which circLMNB1 knockdown inhibits the metastasis of CRC. Finally, this study suggests circLMNB1 as a novel biomarker for CRC.

Tumor microenvironment-responsive fluorogenic nanoprobe for ratiometric dual-channel imaging of lymph node metastasis.

Fluorogenic nanoprobes capable of providing microenvironmental information have extensively been developed to improve the diagnostic accuracy for early or metastatic cancer detection. In cancer-associated microenvironment, matrix metalloproteinase-2,9 (MMP-2,9) has drawn attention as a representative enzymatic marker for diagnosis, prognosis, and prediction of various cancers, which is overexpressed in the primary site as well as metastatic regions. Here, we devised dual-emissive fluorogenic nanoprobe (DFNP) emitting both MMP-2,9-sensitive and insensitive fluorescence signals, for accurate monitoring of the MMP-2,9 activity in metastatic regions. DFNP was nanoscopically constructed by amphiphilic self-assembly between a constantly fluorescent polymer surfactant labeled with Cy7 (F127-Cy7) and an initially nonfluorescent hydrophobic peptide (Cy5.5-MMP-Q) that is fluorogenic in response to MMP-2,9. Ratiometric readout (Cy5.5/Cy7) by dual-channel imaging could normalize the enzyme-responsive sensing signal relative to the constantly emissive internal reference that reflects the probe amount, allowing for semi-quantitative analysis on the MMP-2,9-related tissue microenvironment. In addition to the dual-channel emission, the nanoconstructed colloidal structure of DFNP enabled efficient accumulation to lymph node in vivo. Because of these two colloidal characteristics, when injected intradermally to a mouse model of lymph node metastasis, DFNP could produce reliable ratiometric signals to provide information on the MMP-2,9 activity in the lymph nodes depending on metastatic progression, which corresponded well to the temporal histologic analysis. Furthermore, ratiometric lymph node imaging with DFNP after photodynamic therapy allowed for monitoring a therapeutic response to the given cancer treatment, demonstrating diagnostic and prognostic potential of the nanoconstructed colloidal sensor of tumor microenvironment in cancer treatment.

Mesenchymal stromal cells for bone sarcoma treatment: Roadmap to clinical practice.

Over the past few decades, there has been growing interest in understanding the molecular mechanisms of cancer pathogenesis and progression, as it is still associated with high morbidity and mortality. Current management of large bone sarcomas typically includes the complex therapeutic approach of limb salvage or sacrifice combined with pre- and postoperative multidrug chemotherapy and/or radiotherapy, and is still associated with high recurrence rates. The development of cellular strategies against specific characteristics of tumour cells appears to be promising, as they can target cancer cells selectively. Recently, Mesenchymal Stromal Cells (MSCs) have been the subject of significant research in orthopaedic clinical practice through their use in regenerative medicine. Further research has been directed at the use of MSCs for more personalized bone sarcoma treatments, taking advantage of their wide range of potential biological functions, which can be augmented by using tissue engineering approaches to promote healing of large defects. In this review, we explore the use of MSCs in bone sarcoma treatment, by analyzing MSCs and tumour cell interactions, transduction of MSCs to target sarcoma, and their clinical applications on humans concerning bone regeneration after bone sarcoma extraction.

Landscape of Molecular Events in Pituitary Apoplexy.

Apoplectic pituitary adenomas cause significant morbidity and even mortality. The pituitary apoplexy denotes a pituitary adenoma presenting with hemorrhage and/or infarction, implementation in remedial effects of various of drugs in pituitary apoplexy is a promising pharmacogenomic field in the near future adenoma treatment. Indisputably, this is an important horizon for complicated pituitary adenomas. In a pituitary adenoma, the interplay between genetic, cytokine, and growth factors promotes the pathogenic transformation into an apoplectic formation. However, till date, little is known about how all these factors together lead to the pathogenesis of apoplectic pituitary. The vascular endothelial growth factor, tumor necrosis factor-α (TNF-α), pituitary tumor-transforming gene (PTTG), matrix metalloproteinase-2/9 (MMP-2/9), proliferating marker (Ki-67), as well as hypoxia-inducing factor are the major contributing factors involved in pituitary apoplexy. The molecular mechanism involved in pituitary apoplexy has never been described so far. In this review, we discuss the various proteins/cytokines/growth factors and signaling molecules which are involved in the pathogenesis of pituitary apoplexy and their potential role as biomarkers or as therapeutic targets.

Zinc improves learning and memory abilities of fetal growth restriction rats and promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 axis activity.

Fetal growth restriction (FGR) is a well-known risk factor for cognitive dysfunction, especially for learning and memory abilities. However, knowledge about prevention and treatment methods of learning and memory abilities of fetal are limit. Here, Morris water maze and passive avoidance tests showed zinc supplementation could protect the impairment of the learning and memory abilities caused by FGR. As accumulating evidence suggested that insufficiency of placental trophoblast cell invasion was closely related to FGR fetal neurodevelopmental dysplasia, we further explored the relationship between zinc supplementation during pregnancy and placental trophoblast. Microarray identified 346 differently expressed genes in placental tissues with and without zinc supplementation, and GO and KEGG analyses showed these differently expressed genes were highly enriched in cell invasion and migration and STAT3 pathway. Protein-protein interaction(PPI) analysis found that STAT3 interacted with matrix metalloproteinase-2/9 (MMP-2/9). In vivo, western blot results authenticated that the expression levels of phospho-STAT3, STAT3, MMP-2 and MMP-9 were up-regulated in placental tissues after zinc treatment. To validate whether zinc could promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 activity. In vitro, Transwell assay was performed, and we observed that abilities of invasion and migration were obviously increased in zinc treated trophoblast cells. And phospho-STAT3, STAT3, MMP-2 and MMP-9 expression levels were correspondingly increased in zinc treated trophoblast cells, which were dose-dependent. Moreover, gain-of-function and loss-of-function of STAT3 confirmed that zinc promotes cell invasion and migration via regulating STAT3 mediated up-regulation of MMP-2/9 activity. We propose that activation of MMP-2/9 mediated by STAT3 may contribute to invasion and migration of trophoblast cells, which improved neurodevelopmental impairment of FGR rats probably via contributing to placental development. Our findings are the first to show a possible mechanism of reversing neurodevelopmental impairment of FGR rats by zinc supplementation, holding promise for the development of novel therapeutic modalities for learning and memory abilities impairment caused by FGR.

Honokiol suppresses TNF-α-induced migration and matrix metalloproteinase expression by blocking NF-κB activation via the ERK signaling pathway in rat aortic smooth muscle cells.

Honokiol, a small-molecule polyphenol derived and isolated from the Chinese medicinal herb Magnolia officinalis, has been shown to possess a wide range of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on tumor necrosis factor-α (TNF-α)-induced migration in rat aortic smooth muscle cells (RASMCs). We found that honokiol inhibited TNF-α-induced RASMC proliferation and migration in a dose-dependent manner. At the molecular level, pretreatment with honokiol blocked TNF-α-induced protein expression of matrix metalloproteinase (MMP)-2 and MMP-9, nuclear factor (NF)-κB activation, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, NF-κB inhibitor (BAY 11-7028) and ERK inhibitor (U0126) also mimicked the inhibitory effects of honokiol in TNF-α-treated RASMCs. In conclusion, these results indicate that honokiol suppresses TNF-α-induced migration and MMP expression by blocking NF-κB activation via the ERK signaling pathway in RASMCs. Our findings support honokiol as a promising novel agent for the prevention and treatment of atherosclerosis.

Involvement of the blood-brain barrier opening in cognitive decline in aged rats following orthopedic surgery and high concentration of sevoflurane inhalation.

The underlying causes of postoperative cognitive decline (POCD) in old patients remained unelucidated, and there are little descriptions on mechanisms associated with the blood-brain barrier (BBB) disruption during POCD. We therefore tested the effects of orthopedic surgery with different concentrations of sevoflurane for 2 h on the behavior test and the BBB permeability in aged rats. 18-month rats were divided into control group and surgical group with propofol anesthesia (0.7 mgkg(-1) min(-1)) and 1.0 MAC, 1.3 MAC, and 1.5 MAC sevoflurane inhalation for 2 h. We assessed their cognitive function via Y-maze and fear conditioning test on day 1, 3, and 7 after experiments. Animals were then assigned to control group, propofol (2 h, 0.7 mgkg(-1) min(-1)) group, surgery plus propofol group and surgery plus 1.5 MAC sevoflurane inhalation for 2h. Their hippocampal BBB permeability was detected with Evans blue quantification. Alterations of tight junctions in hippocampus were measured with occludin and claudin-5 western blot. Then we assessed matrix metalloproteinase-2,9 (MMP-2,9) via western blot and immunohistochemistry staining at day 1, 3, 7, and 14 after experiments. Surgery impaired cognitive function and increased Evans blue leakage into the hippocampus in aged rats while 2 h of 1.5 MAC sevoflurane inhalation potentiated these effects. Surgery induced occludin protein expression decreases and MMP-2,9 proteins increase and these influences can be enhanced by high concentration of sevoflurane inhalation. In conclusion, 1.5 MAC sevoflurane for 2 h exacerbated cognitive impairment induced by orthopedic surgery in aged rats and the breach in BBB may be involved in this process.

Correlation of matrix metalloproteinase-2, 9 in gingival crevicular fluid with periodontal inflammation / 西安交通大学学报(医学版)

Objective To examine the changes of matrix metalloproteinase-2,9 (MMP-2,9) in gingival crevicular fluid(GCF) after phase 1 periodontal treatment of adult patients with periodontitis. Methods GCF was sampled with filter paper strips by intra-pocket method to determine MMP-2,9 levels. Forty teeth of forty adult patients with periodontitis and forty teeth of forty periodontally healthy persons were included in this study. Assays for MMP-2,9 in GCF were performd by ELISA. Results Contents of MMP-2,9 were higher in AT group than those in controls(P