Evaluation of significant gene expression changes in congenital and acquired cholesteatoma.

Etiopathogenesis of acquired and congenital cholesteatoma is still unclear. The clinical behavior of adult acquired, pediatric acquired and congenital cholesteatomas show differences. The scope of the this study was to detect the matrix metalloproteinase (MMP), tissue inhibitors of metalloproteinase (TIMP) and epidermal growth factor receptor (EGFR) gene expression changes in cholesteatoma perimatrix and to compare these changes among congenital cholesteatoma, adult acquired cholesteatoma and pediatric acquired cholesteatoma. A total of 16 genes including MMPs, TIMPs and EGFR were analyzed in the samples of 32 cholesteatoma tissues. Real-time PCR was used for detection of the gene expression levels. Data analyses were achieved by ΔΔCT method (Light Cycler 480 Quantification Software) and Statistical Package for Social Sciences (SPSS) version 22.0. The expression levels of MMP-2, -9, -10, -11, -13, -14, -15, -16 and EGFR genes were significantly higher in acquired cholesteatoma than healthy tissue (p < 0.05). There was a statistically significant decrease (3.34 times more) in the mean TIMP-2 gene expression level in acquired cholesteatoma compared to healthy tissue (p < 0.05). There was a significant increase in the mean expression level of MMP-7 gene and a decrease in the mean expression level of TIMP-1 gene (3.12 times more) in congenital cholesteatoma compared to healthy tissue (p < 0.05). This study indicates that increased expression levels of some particular MMP genes and EGFR gene and decreased expression levels of TIMP genes may play an important role in the development of cholesteatoma. Further, MMP-9, MMP-13 and MMP-14 genes may have a remarkable role in the development of more aggressive cholesteatoma forms. The authors concluded that overexpression of MMP-9, MMP-13 and MMP-14 may cause stronger inflammation associated with cholesteatoma.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during maturation.

Although participation of matrix metalloproteinases (MMPs) in reproductive tract remodeling has been strongly suggested in mammalian species, the role of MMPs in the avian oviduct has received little attention. To gain a better understanding of the potential role of the MMP system in avian oviduct development, mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and gelatinolytic activity in the oviduct of growing chickens were examined. The oviducts were collected from Hy-Line Brown hens before (10, 12, 14 and 16 weeks of age) and after (week 17) the onset of egg laying. The MMP-2, -7, -9 and TIMP-2 and -3 genes were found to be differentially expressed in all examined oviductal sections: the infundibulum, magnum, isthmus and shell gland on both mRNA (by real time polymerase chain reaction) and protein (by western blotting and immunohistochemistry) levels. In the course of oviduct development, the relative expression of all genes decreased in most sections. Protein level of MMP-9 was diminished, while MMP-7 and TIMP-3 were elevated in the oviduct of growing birds. MMP-2 and TIMP-2 protein levels remained constant, with a slight increase in MMP-2 concentration just before reaching maturity. The relative activity of MMP-2 and -9 (assessed by gelatin zymography) was higher (P < 0.05, P < 0.01) in immature birds compared with adults. Immunohistochemistry demonstrated cell- and tissue-specific localization of MMPs and TIMPs in the wall of the chicken oviduct. We concluded that changes in the expression of examined MMPs and their inhibitors, as well as alterations in MMP activity occurring simultaneously with changes in the morphology of the chicken oviduct, suggest the involvement of the MMP system in the proper development and functioning of this organ. Mechanisms regulating the expression and activity of MMPs require further clarification.

Tumor progression driven by pathways activating matrix metalloproteinases and their inhibitors.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is still a problem worldwide. In some publications interactions between the expression of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and their tissue inhibitors (TIMPs) implicated during cancer progression were suggested. METHODS: The immunohistochemical staining using primary antibody against MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were performed. The research group consists of primary N(0) LSCC (20 cases), primary N(+) LSCC (17 cases), and 18 cases of normal mucosa. RESULTS: Studied MMPs and TIMPs were localized in tumor cells and tumor stroma compartment. MMP-2 expression was higher in stroma compared to tumor cells. MMP-9, TIMP-1, TIMP-2, and TIMP-3 expression was higher in tumor cells than in tumor stroma (P < 0.05). In tumor stroma MMP-2, MMP-9, TIMP-1, and TIMP-3 expression, in LSCC N(0) vs. LSCC N(+) was significantly higher (P < 0.05). The ratios between MMP-2 and TIMP-3 expression were statistically significant (N(0) vs. N(+); P = 0.012). The analyses using classification trees predicted the probability of metastases according to TIMP-3/MMP-14/MMP-2 and MMP-9/TIMP-1 expression levels. CONCLUSIONS: The presence of MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3 expression in tumor cells and in tumor stroma, and additionally different expression according to lymph node involvement suggested of their impact during cancer progression. The significant correlation between TIMP-3 expression and the presence of lymph node metastases and MMP-2 expression might suggest the importance of TIMP-3 as a prognostic factor during tumor progression. The evaluation of molecular markers which participate in MMP-2 activation pathway have a major impact during metastasis.

Identification of shoulder osteoarthritis biomarkers: comparison between shoulders with and without osteoarthritis.

BACKGROUND: The biologic factors associated with shoulder osteoarthritis (OA) have not been elucidated. The purpose of this study was to investigate osteoarthritic biomarkers of the shoulder. To our knowledge, this is the first study to analyze shoulder cartilage for OA-associated genes and to examine human shoulder cartilage for a possible biomarker, connexin 43 (Cx43). MATERIALS AND METHODS: Cartilage from 16 osteoarthritic and 10 nonosteoarthritic humeral heads was assessed for expression of the following genes by real-time polymerase chain reaction: types I, II, and X collagen; matrix metalloproteinases (MMPs); tissue inhibitors of MMP (TIMPs); interleukins; versican; cyclooxygenase 2 (Cox-2); inducible nitric oxide synthase (iNOS); tumor necrosis factor α (TNF-α); aggrecanase 2 (ADAMTS5); and Cx43. RESULTS: In osteoarthritic shoulders, Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expressions were significantly increased compared with controls. TIMP-3 and iNOS trended toward significance, with robust expression in osteoarthritic shoulders and low expression in nonosteoarthritic shoulders. In osteoarthritic shoulders, gene expression of Cx43, ADAMTS5, collagen type I, Cox-2, versican, and TIMP-3 showed predominance (85-, 33-, 13-, 12-, 11.5-, and 3-fold increases, respectively) relative to nonosteoarthritic controls. Spearman correlation analysis showed significant correlations between Cx43 and collagen (types I, II, and X), MMP-9, TIMP-2 and TIMP-3, versican, Cox-2, iNOS, and ADAMTS5. CONCLUSIONS: Certain genes are markedly upregulated in osteoarthritic shoulders compared with nonosteoarthritic shoulders, with Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expression being significantly increased. These genes might be useful biomarkers for examining shoulder OA. CLINICAL RELEVANCE: Identification of osteoarthritic biomarkers can help us better understand shoulder OA and build the foundation for future research on disease progression and treatments.

Epigenetic reprogramming reverses the malignant epigenotype of the MMP/TIMP axis genes in tumor cells.

Cancer progression is characterized by extensive tumor invasion into the surrounding extracellular matrix (ECM) and migration to metastatic sites. The increased proteolytic degradation of the ECM during tumor invasion is directly dependent on the activity of matrix metalloproteinases (MMPs), counter-balanced by tissue inhibitors of matrix metalloproteinases (TIMPs). In this study, we found that unbalanced expression of MMP/TIMP axis genes in tumors was correlated with aberrant epigenotypes in the various gene promoters. The malignant epigenotypes could be therapeutically corrected by a simple defined factor-mediated reprogramming approach. Correction of the abnormal epigenotypes by nuclear remodeling leads to a rebalance in the gene expression profile, an alteration in tumor cell morphology, attenuation of tumor cell migration and invasion in vitro, and reduced tumorigenicity in nude mice. We further identified the downregulation of the MKK-p38 MAPK signal pathway as an important underlying mechanism for reduced tumorigenicity in this epigenetic reprogramming model. These data demonstrate that the malignant phenotypes seen in cancer can be corrected by a nuclear remodeling mechanism, thus highlighting a novel non-chemotherapeutic, non-radiotherapeutic approach for the treatment of cancer.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1ß induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes.

OBJECTIVE: Interleukin-1ß (IL-1ß) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS: Effects of WIN-55 were studied on IL-1ß stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS: Treatment with WIN-55 alone or in combination with IL-1ß, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1ß, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION: Cannabinoid WIN-55 can reduce both basal and IL-1ß stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

[The influence of risk factors for malignant tumors of maxillo-facial area on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in patients of elderly and senile age period].

Morbidity and mortality from malignant neoplasms increases each year, and the average age of patients with first diagnosed decreases. The peak of incidence of malignant tumors of maxillofacial region falls on the elderly life. Not only age-related changes of the organism contribute in the development of this pathology, but also harmful habits. A comprehensive approach to solving the problem requires monitoring of this group of patients to identify risk factors for the development of pathologies in the oral and maxillofacial region.

Suppression of plasminogen activators and the MMP-2/-9 pathway by a Zanthoxylum avicennae extract to inhibit the HA22T human hepatocellular carcinoma cell migration and invasion effects in vitro and in vivo via phosphatase 2A activation.

This study shows that the ECM degradation-associated pathway, including uPA and tPA and the downstream MMP-2/-9 protein, was significantly suppressed in HA22T cells treated with a Zanthoxylum avicennae extract (YBBE). The endogenous inhibitors, including TIMP-1/-2 and PAI-1, were enhanced in HA22T cells by the YBBE treatment. The expression of MMP-2/-9 and TIMP-1/-2 was respectively assessed by using RT-PCR and a zymography assay. The mRNA levels and enzymatic activity of MMP-2/-9 were down-regulated by the YBBE treatment in a dose-dependent manner, while the TIMP-1/-2 levels were conversely markedly increased. The PP2A siRNA or PP2A inhibitor totally reversed the YBBE effects, confirming the essential role of PP2A in YBBE inhibiting the HA22T cell migration and invasion effects. Xenografted animal experiments on nude mice demonstrated similiar results to the in vitro system. Both the in vitro and in vivo models clearly demonstrate that YBBE inhibited the highly metastatic HA22T liver cancer cell migration and invasion effects through PP2A activation.

Minocycline suppresses experimental autoimmune encephalomyelitis by increasing tissue inhibitors of metalloproteinases.

Matrix metalloproteinases (MMPs) that are secreted by activated T cells play a significant role in degradation of the extracellular matrix around the blood vessels and facilitate autoimmune neuroinflammation; however, it remains unclear how MMPs act in lesion formation and whether MMP-targeted therapies are effective in disease suppression. In the present study, we attempted to treat experimental autoimmune encephalomyelitis (EAE) by administration of small interfering RNAs (siRNAs) for MMP-2, MMP-9, and minocycline, all of which have MMP-inhibiting functions. Minocycline, but not siRNAs, significantly suppressed disease development. In situ zymography revealed that gelatinase activities were almost completely suppressed in the spinal cords of minocycline-treated animals, while significant gelatinase activities were measured in the EAE lesions of control animals. However, MMP-2 and MMP-9 mRNAs and proteins in the spinal cords of treated rats were unexpectedly upregulated. At the same time, mRNA for tissue inhibitors of MMPs (TIMP)-1 and -2 were also upregulated. The EnzChek Gelatinase/Collagenase assay using tissue containing native MMPs and TIMPs demonstrated that gelatinase activity levels in the spinal cords of treated rats were suppressed to the same level as those in normal spinal cord tissues. Finally, double immunofluorescent staining demonstrated that MMP-9 immunoreactivities of treated rats were almost the same as those of control rats and that MMP-9 and TIMP-1 immunoreactivities were colocalized in the spinal cord. These findings suggest that minocycline administration does not suppress MMPs at mRNA and protein levels but that it suppresses gelatinase activities by upregulating TIMPs. Thus, MMP-targeted therapies should be designed after the mechanisms of candidate drugs have been considered.

Matrix metalloproteinases and their inhibitors in squamous cell carcinoma of the conjunctiva.

Squamous cell carcinoma of the conjunctiva (SCCC) belongs to a disease spectrum known as ocular surface squamous neoplasia (OSSN). Epidemiological evidence suggests that environmental ultraviolet radiation (UVR) exposure is the principal triggering agent. Despite this indirect evidence, the pathogenesis of SCCC remains poorly understood. We postulated that matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are upregulated in SCCC, and this could account for the invasive activity associated with this disease. Archival tissue specimens from 10 patients with SCCC were acquired to assess the expression of seven MMPs, three TIMPs, and two growth factors and their receptor by immunohistochemistry using specific antibodies. All MMPs and TIMP-2 were overexpressed in the tumor component compared to adjacent normal conjunctiva and cornea. Active MMP-7 was detected in diseased tissue, suggesting that at least some members of this family of enzymes are functionally involved. Moreover, active epidermal growth factor receptor (EGFR) and its ligands were detected within the tumor compartment. These data suggest that UVR-induced downstream cellular signaling events, including activation of cell-surface receptors and the induction of downstream effector molecules, such as MMPs and growth factors, are involved in the pathogenesis of SCCC. Mapping and inhibiting these pathways may aid in delineating the pathogenesis of SCCC and provide clues for optimizing current therapeutic methods or developing novel treatment strategies.

Elevation of matrix metalloproteinases in different areas of ascending aortic aneurysms in patients with bicuspid and tricuspid aortic valves.

Our aim is to investigate the elevation of matrix proteins in tissues obtained from distal, above the sinotubular junction (proximal), concave, and convex sites of aneurysms in the ascending aorta using a simultaneous multiplex protein detection system. Tissues were collected from 41 patients with ascending aortic aneurysms. A total of 31 patients had a bicuspid aortic valve (BAV), whereas 10 had a tricuspid aortic valve (TAV). Concave and convex aortic site samples were collected from all patients, whereas proximal and distal convexity samples were obtained from 19 patients with BAV and 7 patients with TAV. Simultaneous detection of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) was performed at each of the four aortic sites. MMP-2 levels were higher in the concave aortic sites than in the convex aortic sites. In contrast, MMP-8 levels were higher in the convex sites than in the concave sites, as were MMP-9 levels. In both BAV and TAV patients, TIMP-3 levels were higher in the concave sites than in the convex sites. However, TIMP-2 and TIMP-4 levels were significantly elevated in the sinotubular proximal aorta of BAV patients. Simultaneous detection of MMPs and TIMPs revealed different levels at different aortic sites in the same patient.

Matrix metalloproteinases and their inhibitors and inducer in gestational trophoblastic diseases and normal placenta.

OBJECTIVES: This study aimed to investigate the expression of recently identified matrix metalloproteinases (MMPs), their inhibitors (TIMPs), and inducer (CD147) in a wide range of gestational trophoblastic diseases (GTD) thereby expanding our understanding of the potential role of MMPs in GTD. METHODS: Paraffin sections of 10 normal first-trimester placentas (NP), 10 partial moles (PM), 10 complete moles (CM), 5 choriocarcinomas (CCA) and 5 placental site trophoblastic tumors (PSTT) were studied immunohistochemically for expression of MMP-7, MMP-14, MMP-21, MMP-28, TIMP-3, TIMP-4 and CD147. Immunolocalization of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and TIMP-1 was performed on 5 CCA and 5 PSTTs. RESULTS: CCA showed stronger intensity for MMP-14 and MMP-28 than PSTT (p<0.05, p<0.05). CCA and PSTT had stronger expression of MMP-21 than NP, PM and CM (p<0.05, p<0.05, p<0.01). PSTT (p<0.05, p<0.05), NP (p<0.01, p<0.01) and CM (p<0.01, p<0.05) showed stronger staining for TIMP-3 and TIMP-4 than CCA. CONCLUSION: Choriocarcinoma's high expression of MMPs and low expression of MMP inhibitors may contribute to its invasiveness and metastatic potential. Similarly, PSTT's lower expression of MMPs and high expression of MMP inhibitors may partly explain its lower invasiveness. Agents that inhibit MMP may prove useful in treating GTD.

Collagenase-3 expression by tumor cells and gelatinase B expression by stromal fibroblast-like cells are associated with biochemical recurrence after radical prostatectomy in patients with prostate cancer.

PURPOSE: To investigate the possible clinical value of the expression of MMPs and their tissue inhibitors (TIMPs) by the different cellular types of the tumor scenario to predict biochemical recurrence in patients undergoing radical prostatectomy due clinically localized prostate cancer. METHODS: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs-1, 2, 7, 9, 11, 13 and 14 and TIMPs-1, 2 and 3 on cancer specimens from 133 patients with clinical localized prostate carcinoma. RESULTS: Immunostaining for all the proteins studied was localized predominantly in tumor cells, but also in stromal cells in a significant percentage of prostate carcinomas, ranged from 20 to 50% for several proteins in fibroblast-like cells and in mononuclear inflammatory cells. Multivariate analysis according to a Cox model demonstrated that tumor stage (P < 0.0001) and Gleason grading (grades 7-10: 2.08 (1.1-3.9); P < 0.05) were significantly and independently associated with biochemical recurrence. Additionally, the expression of MMP-9 by fibroblast-like cells (P < 0.01) and MMP-13 by tumor cells (P < 0.05) were also variables significantly and independently associated with biochemical recurrence. CONCLUSIONS: MMP-13 expression by tumor cells and MMP-9 by stromal fibroblast-like cells were independent factors of biochemical recurrence in prostate cancer.

Effects of propofol on the expression of matric metalloproteinases in rat cardiac fibroblasts after hypoxia and reoxygenation.

BACKGROUND: Propofol is known to protect the myocardium against ischaemia/reperfusion (I/R) injury through its antioxidant and anti-inflammatory properties. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in cell migration and invasion, and mediate tissue remodelling during I/R injury. They are regulated by various mechanisms including oxidative stress and AKT and ERK pathways. We investigated whether propofol affected the expression of MMPs and subsequent cell migration and invasion and the signalling pathways involved in primary rat cardiac fibroblasts undergoing hypoxia and reoxygenation. METHODS: The phosphorylation of ERK and AKT signalling pathways was examined by western blot analysis in rat primary cardiac fibroblasts after hypoxia and reoxygenation. mRNA expression of MMP and TIMPS was analysed by real-time PCR, and proteolytic activities of MMP-2 and -9 were assessed. The effects of propofol on migration, invasion, wound healing, and cell proliferation activity were evaluated after reoxygenation. RESULTS: Propofol induced AKT and ERK1/2 activation. Subsequent activation of MMPs resulted in increased cell migration, invasion, and wound-healing activity under hypoxia-reoxygenation, which was decreased by LY294002 (AKT inhibitor) and U0126 (ERK inhibitor) in rat cardiac fibroblasts. However, propofol had no effect on proliferation or viability of cardiac fibroblasts after hypoxia-reoxygenation. CONCLUSIONS: Propofol affected the expression of MMPs and TIMPs and subsequently induced cell migration and invasive ability, through activation of the ERK and AKT signalling pathway in hypoxia-reoxygenated rat cardiac fibroblasts.

Granulocyte-macrophage colony-stimulating factor initiates amniotic membrane rupture and preterm birth in a mouse model.

OBJECTIVE: Preterm premature rupture of membranes is associated with 30% of all preterm births. The weakening of amniotic membranes is associated with an increase in matrix metallopeptidases (MMPs) along with a decrease in their inhibitors, tissue inhibitor metallopeptidases (TIMPs). Additionally, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to weaken fetal membranes in-vitro. We hypothesize pregnant mice treated with GM-CSF lead to increased MMPs:TIMPs resulting in membrane rupture and preterm birth. STUDY DESIGN: Pregnant CD-1 mice on gestational day 17 received either an intrauterine injection of GM-CSF or vehicle control. A second series of mice were administered an intrauterine injection of Lipopolysaccharide along with either anti-mouse GM-CSF or control antibody. Mice were evaluated for rupture of membranes and/or preterm birth and the uterus, amniotic fluid, and serum were collected for analysis. RESULTS: 87.5% of GM-CSF mice exhibited evidence of membrane rupture or preterm birth, compared with 0% in control mice (p < .001). Treatment with GM-CSF decreased the expression of TNFα (p < .05) while increasing the ratio of MMP2:TIMP1 (p < .05), MMP2:TIMP2 (p < .05), MMP2:TIMP3 (p < .001), MMP9:TIMP1 (p < .01), MMP9:TIMP2 (p < .05), MMP9:TIMP3 (p < .001), and MMP10:TIMP1 (p < .05). Mice treated with LPS and the GM-CSF antibody resulted in a decrease in the ratio of MMP2:TIMP1 (p < .0001) compared with controls. CONCLUSION: These studies demonstrate GM-CSF will result in membrane rupture and preterm birth by increasing the ratio MMPs:TIMPs in our animal model. By increasing our understanding of the molecular pathways associated with GM-CSF, we may be able to develop future therapies to prevent preterm birth and reduce neonatal morbidity.

TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas.

Based on the molecular profiling of astrocytomas, we previously identified a series of genes involved in astrocytoma invasion. Of these, tissue inhibitor of metalloproteinase-4 (TIMP-4) was found to be overexpressed in pilocytic astrocytomas relative to diffuse astrocytomas of any histological grade. Although some data suggest that TIMP-4 may be an anti-tumoral actor in astrocytomas, recent findings challenge this concept. The present study aims to investigate the diagnostic and prognostic values of TIMP-4 and its putative partner CD63 in human astrocytomas. Tissue microarray and image analysis were first carried out to quantitatively analyze the immunohistochemical expression of these proteins in 471 gliomas including 354 astrocytomas. Pathological semi-quantitative scores of both markers' expression were then established and correlated to astrocytoma diagnosis and patient prognosis. TIMP-4 and CD63 expressions were both overexpressed in astrocytomas compared with oligodendrogliomas (P<0.001) and in pilocytic astrocytomas compared with grade II diffuse astrocytomas (P<0.001). In glioblastomas, high TIMP-4/CD63 co-expression scores were identified as independent prognostic factors associated with progression and shorter survival. In conclusion, this work provides the first evidence of a TIMP-4/CD63 association in astrocytoma tumor cells. It identifies TIMP-4 and CD63 as markers of the astrocytic phenotype in patients with gliomas. In addition, this work highlights the contribution of high TIMP-4/CD63 co-expression to the adverse outcomes of patients with glioblastomas.

Tissue inhibitor of metalloproteinase-4 is elevated in early-stage breast cancers with accelerated progression and poor clinical course.

An increasing number of breast cancer patients are diagnosed with small, localized, early-stage tumors. These patients are typically thought to have a good prognosis for long-term disease-free survival, but epidemiological studies indicate that up to 30% may have a recurrence within 3 to 5 years of diagnosis. Identifying patients with a high risk of recurrence and/or progression is important because they could be more aggressively treated at diagnosis to improve their chances for disease-free survival. Recent evidence suggests that elevated levels of the matrix metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP)-4, are associated with malignant progression of ductal carcinoma in situ, a precancerous lesion. To examine the association of TIMP-4 with survival outcomes, we conducted a retrospective immunohistochemical analysis of 314 cases from patients with early-stage disease, defined as tumors smaller than 2 cm and no spread to lymph nodes (tumor-node-metastasis staging: T1N0MX). We found that tumors with elevated levels of TIMP-4 were correlated with a reduced probability of long-term disease-free survival, especially in patients with estrogen receptor-negative tumors. Our findings prompt further evaluation of TIMP-4 as a simple prognostic marker that may help identify patients with early-stage breast cancer who could benefit from more aggressive treatment at diagnosis.

The effect of oxygen tension on the long-term osteogenic differentiation and MMP/TIMP expression of human mesenchymal stem cells.

The use of mesenchymal stem cells in tissue engineering to augment the repair of a variety of tissues including bone is a rapidly growing and exciting field. Although oxygen tension is a powerful stimulus for cells both in vitro and in vivo, the oxygen environment in which such cells would undergo differentiation is commonly overlooked. We examined the effect of long-term (21-days) low oxygen tension (1, 2 and 5%) on the osteogenic differentiation and matrix metalloproteinase (MMP)/tissue inhibitor of MMP (TIMP) expression of human mesenchymal stem cells (MSCs). Our data suggest that MSCs undergo osteoblastic differentiation most rapidly under 21% oxygen while oxygen tensions below 5% have an inhibitory effect. Interestingly, there was not a statistically significant difference in osteogenic markers between 5 and 21% oxygen. In addition, our data suggest that oxygen tension affects the expression of individual MMP and TIMPs differently. Low oxygen tension has an inhibitory effect on MMP-13 and TIMP-1 expression, which are involved in extracellular matrix remodeling and potentially vascular invasion. In contrast, MMP-2, a metalloproteinase involved in cell migration was not affected by oxygen tension. This data suggests that 21% oxygen may be beneficial for rapid osteogenic differentiation as would be required for the production of individual patient ex vivo constructs. In addition, this has important in vivo implications relating to the importance of early vascularization of sites of orthopedic injury. By augmenting the neovascularization process, it may be possible to facilitate more rapid differentiation of progenitors and thus the repair process.

Matrix metallopeptidase expression and modulation by transforming growth factor-ß1 in equine endometrosis.

Equine endometrial fibrosis (endometrosis) is described as a degenerative chronic condition in the uterus. Its characteristic feature is excessive deposition of extracellular matrix (ECM) components around the endometrial glands and stroma. Although matrix metallopeptidases (MMPs) that mediate ECM turnover are important factors in the process of fibrosis, knowledge of their expression and regulation in endometrosis is limited. In other species, one of the important regulators of MMPs and tissue inhibitors of MMPs (TIMPs) is transforming growth factor (TGF)-ß1. The goal of this study was to determine (i) endometrial expression of MMPs and TIMPs during endometrosis and (ii) the effect of TGF-ß1 on expression of MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells. In the follicular phase of the estrous cycle, MMP-1, -2, -9, and TIMP concentrations were higher during endometrosis than in healthy endometrium (P < 0.05). In the midluteal phase, MMP-3 concentration was lower in severe endometrosis compared to healthy endometrium (P < 0.05). In fibroblasts, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP1, but downregulated MMP-3 secretion (P < 0.05). In epithelial cells, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP secretion (P < 0.05). Endometrial expression of MMPs and TIMPs is altered during endometrosis. TGF-ß1 is a regulator of endometrial ECM remodeling via its effect on MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells.

1alpha,25-dihydroxyvitamin D3 inhibits matrix metalloproteinases induced by Mycobacterium tuberculosis infection.

Matrix metalloproteinases (MMP) can degrade all components of pulmonary extracellular matrix. Mycobacterium tuberculosis induces production of a number of these enzymes by human macrophages, and these are implicated in the pathogenesis of pulmonary cavitation in tuberculosis. The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], has previously been reported to inhibit secretion of MMP-9 in human monocytes (MN), but its influence on the secretion and gene expression of MMP and tissue inhibitors of MMP (TIMP) in M. tuberculosis-infected cells has not previously been investigated. We therefore determined the effects of 1alpha,25(OH)(2)D(3) on expression, secretion and activity of a number of MMP and TIMP in M. tuberculosis-infected human leucocytes; we also investigated the effect of 1alpha,25(OH)(2)D(3) on the secretion of interleukin-10 (IL-10) and prostaglandin E(2) (PGE(2)), both transcriptional regulators of MMP expression. We found that M. tuberculosis induced expression of MMP-1, MMP-7 and MMP-10 in MN and MMP-1 and MMP-10 in peripheral blood mononuclear cells (PBMC). 1alpha,25(OH)(2)D(3) significantly attenuated M. tuberculosis-induced increases in expression of MMP-7 and MMP-10, and suppressed secretion of MMP-7 by M. tuberculosis-infected PBMC. MMP-9 gene expression, secretion and activity were significantly inhibited by 1alpha,25(OH)(2)D(3) irrespective of infection. In contrast, the effects of 1alpha,25(OH)(2)D(3) on the expression of TIMP-1, TIMP-2 and TIMP-3 and secretion of TIMP-1 and TIMP-2 were small and variable. 1alpha,25(OH)(2)D(3) also induced secretion of IL-10 and PGE(2) from M. tuberculosis-infected PBMC. These findings represent a novel immunomodulatory role for 1alpha,25(OH)(2)D(3) in M. tuberculosis infection.