Search

Purpose: The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFβ) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFβ-mediated PCO formation., Methods: The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models., Results: Active TGFβ2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFβ2-induced matrix contraction. Active TGFβ2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 μM), MMP2 siRNA, and MMP2 neutralizing antibody (4 μg/mL). TGFβ2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 μg/mL)., Conclusions: MMP2 plays a critical role in TGFβ2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders.

The original notion that matrix metalloproteinases (MMPs) act as tumour and metastasis-promoting enzymes by clearing a path for tumour cells to invade and metastasize has been challenged in the last decade. It has become clear that MMPs are involved in numerous steps of tumour progression and metastasis, and hence are now considered to be multifaceted proteases. Moreover, more recent experimental evidence indicates that some members of the MMP family behave as tumour-suppressor enzymes and should therefore be regarded as anti-targets in cancer therapy. The complexity of the pro- and anti-tumorigenic and -metastatic functions might partly explain why broad-spectrum MMP inhibitors failed in phase III clinical trials. This review will provide a focussed overview of the published data on the tumour-suppressive behaviour of MMPs., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

A 4-component antibody-phthalocyanine-polyethylene glycol-gold nanoparticle conjugate is described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles (4 nm) were stabilised with a self-assembled layer of a zinc-phthalocyanine derivative (photosensitiser) and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently bound to the nanoparticles via a terminal carboxy moiety on the polyethylene glycol. The nanoparticle conjugates were stable towards aggregation, and under irradiation with visible red light efficiently produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.

The human ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) family of 19 secreted, multidomain proteolytic enzymes is involved in a wide range of biological processes including ECM assembly and degradation, hemostasis, organogenesis and the regulation of angiogenesis. Defects in certain family members give rise to inherited human genetic diseases, while aberrant expression of other ADAMTSs has been linked to the pathogenesis of arthritis and cancer. Several ADAMTSs act as tumor or metastasis suppressors whose functions are lost either by mutation or epigenetic silencing during tumor progression. This review looks in depth at the involvement of ADAMTSs as positive and negative mediators in cancer growth and spread.

Purpose: The ADAMs (a disintegrin and metalloproteinase) and the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin-like motifs) are extracellular proteases that mediate cellular interactions and cell signaling via the modulation of adhesion and the cleavage of cell surface protein ectodomains and extracellular matrix molecules. Gene expression profiling was undertaken to better understand the role of the ADAM and ADAMTS families in the clear native human lenses and following surgical injury with particular relevance to posterior capsule opacification., Methods: To carry out profile analysis, the lens (t=0d) was dissected into three regions; anterior epithelia, equatorial region, and fiber cells. Capsular bag culture was undertaken as a means of assessing short-term changes (t=6d) and post-cataractous lens capsular bags (ex vivo) were used to predict long-term changes in ADAM/ADAMTS gene expression. RNA was isolated and quantitative real-time (TaqMan) reverse transcription-PCR (RT-PCR) performed. Data were analyzed in terms of cycle threshold number (C(T)) and also normalized relative to endogenous 18S rRNA., Results: High expression of ADAM-9, -10, -15, and -17 was detected in all native lens regions. ADAM-15 expression was a characteristic of the native lens epithelia more than the fibers. Post-surgical injury, lens capsular bags showed a positive shift in ADAM/ADAMTS expression that was significant for ADAM-9, -15, and ADAMTS-3. Ex vivo capsular bags, with a long-term post surgical injury period, maintained high expression of ADAM-9 and -10 genes., Conclusions: The native human lens expresses ADAM and ADAMTS genes that are differentially regulated following surgical injury. Roles in maintaining cellular adhesion may be of particular importance to native lens tissue integrity and may be lost in the lens wound healing response following cataract surgery.

Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.

Quantitative reverse transcriptase polymerase chain reaction enables the accurate quantification of gene expression in cultured cells or small tissue samples. In this chapter, we describe the use of Taqman((R)) technology to measure expression of matrix metalloproteinases and related genes.

Objective: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 in hyperlipidemic mice affects atherogenesis and aortic dilation., Methods and Results: We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2., Conclusions: When overexpressed in the heart and plasma, TGF-beta1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.

Prostate cancer cells exist under hypoxic conditions. Hypoxia has a detrimental effect on the efficacy of treatment and final outcome in patients with prostate cancer. There have been a large number of endogenous markers of hypoxia described previously across a range of cancer types, both in vitro and in vivo. The aim of this study was to evaluate the expression of a range of hypoxia-associated genes within benign prostatic hypertrophy (BPH) and prostate cancer tissue. Messenger RNA was extracted from primary prostate tissue obtained from 67 men with benign prostatic hypertrophy or prostate cancer (Gleason score 5 to 10). Real-time polymerase chain reaction was performed to quantify the expression levels of 12 hypoxia-associated genes in these tissues. Expression of lysyl oxidase (LOX) and glucose transporter-1 (GLUT-1) genes were significantly higher in prostate cancer compared with BPH tissue (P<0.05) and correlated with Gleason score (LOX: R=0.297, P=0.015; GLUT-1: R=0.274, P=0.026). HIF-2alpha had a negative correlation with Gleason score (R= -0.309, P=0.012). The remaining hypoxia-associated genes did not show any specific pattern of expression in prostate tissue. Numerous molecules have been proposed as endogenous markers of hypoxia. The findings of this study illustrate that not all hypoxia-associated molecules are relevant to prostate cancer in vivo. However, LOX and GLUT-1 are candidate markers of hypoxia in prostate cancer and may prove useful in identifying patients with hypoxic prostate cancer. Not all hypoxia-associated molecules are relevant in prostate cancer in vivo.

Objectives: To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements., Materials and Methods: In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization., Results: Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues., Conclusions: These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.

Selenium is considered to be one of the most promising micronutrients for cancer prevention and therapy, based on evidence from epidemiological studies, laboratory-based research and clinical trial intervention. There are ample reports of selenium methionine and sodium selenite's ability to induce apoptosis in various cancers in vitro. There are a few reports in the literature on the effects of selenium on established glioma cell lines but none on biopsy-derived short-term brain tumour cultures. In this in vitro study the effects of a range of concentrations (2-10 microg/ml) of sodium selenite were investigated in one low-passage culture of biopsy-derived glioma cells (IPSB-18, an anaplastic astrocytoma, P 18-22) and a normal human brain cell culture (CC2565, P11). Results from 2 viability assays, 3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulphorodamine B (SRB) consistently showed that the IC50 for selenium in the astrocytoma was approximately 5 microg/ml whilst the normal brain cells were unaffected by selenium in the range of concentrations studied. Time-lapse video microscopy revealed that, while at 4 microg/ml selenium, the time taken to achieve 100% cell death was 17 h, with increasing concentrations of selenium from 6 to 8 microg/ml and finally at 10 microg/ml the IPSB-18 cells rounded up and died much more quickly. The time taken to achieve 100% cell death was 7 h, 7 h and 6 h, respectively, suggesting that the effect was similar at higher concentrations. Flow cytometry indicated that cell death was by apoptosis. RT-PCR results showed downregulation of the gene expression of 6 matrix metalloproteases (MMP2, 9, 14, 15, 16, 24), their inhibitors, TIMPs and epidermal growth factor receptor, in IPSB-18 cells treated with 2, 4 and 8 microg/ml of selenium. Collectively, the data in this study suggests that selenium, not only induces tumour cell-specific apoptosis but also has anti-invasive potential.

Microglia are resident immune cells within the central nervous system (CNS). They become activated following neurological insults and increase their expression of cytokines. Also elevated in CNS injuries are proteases, including matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs). The spectrum of metalloproteinase members expressed by microglia and by the systemic leukocytes that infiltrate the injured CNS is unknown, as are their functions. We determined the levels of transcripts encoding all 24 MMPs, nine ADAMs, and their four physiological antagonists, tissue inhibitor of metalloproteinases (TIMPs), in human microglia, B and T cells, monocytes, and neutrophils. We found a distinct pattern for each immune subset and an enrichment of metalloproteinases in microglia compared with leukocytes. When microglia were activated, there was an upregulation of transcripts for nine metalloproteinases, and reduction of TIMP3. Activation of microglia also resulted in increased levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 protein in the conditioned media of cells. The amount of secreted TNF-alpha, but not IL-1beta or IL-10, was suppressed by BB94, a broad spectrum metalloproteinase inhibitor, and by TIMP3 but not TIMP1 or TIMP2. This inhibitory profile suggests the involvement of an ADAM member in TNF-alpha secretion. We conclude that microglia bear a metalloproteinase signature distinct from systemic cells, and that following activation, microglia upregulate TNF-alpha protein levels through a combination of elevated cytokine transcripts, increased metalloproteinase level and activity, and through the decrease of TIMP3. The results have implications for the regulation of neuroinflammation and its outcomes following CNS injuries., ((c) 2007 Wiley-Liss, Inc.)

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. Some secreted matrix metalloproteinases (MMPs) as MMP2 are highly upregulated in IPF lungs. Membrane-type (MT)-MMPs participate in the activation of pro-MMP2. However, they have not been examined in IPF., Methods: Type I transmembrane MT-MMPs, MT1, MT2, MT3, and MT5-MMP were analyzed by real-time PCR and immunohistochemistry in IPF and normal lungs. MMP-2 was also immunolocalized and evaluated by gelatin zymography in BAL fluids. Additionally, the MT-MMPs were examined by real time PCR in lung fibroblasts stimulated with TGF-beta1 and IFN-gamma., Results: MT1-MMP, was the most highly expressed followed by MT2- and MT5-MMP, and by a moderate expression of MT3-MMP. Regarding their localization, MT1- and MT2-MMPs were found in alveolar epithelial cells, MT3-MMP in fibroblasts from fibroblastic foci and alveolar epithelial cells and MT5-MMP in basal bronchiolar epithelial cells and in areas of squamous metaplasia. MMP2 was localized in alveolar and basal bronchiolar epithelial cells and fibroblasts, and increased active enzyme was observed in BAL fluids. In lung fibroblasts, TGF-beta1 induced a strong upregulation of MT3-MMP, both at the gene and protein level. This effect was blocked by genistein, a protein tyrosin kinase inhibitor and partially repressed by SB203580 a p38 MAP kinase inhibitor. IFN-gamma had no effect., Conclusions: MT-MMPs are expressed in IPF, in the same cell types as MMP2. Mostly by different types of epithelial cells a pivotal component in the aberrant remodeling of the lung microenvironment. Interestingly MT3-MMP that was found in fibroblastic foci was upregulated in vitro by TGF-beta1 a potent profibrotic mediator.

Objective: To profile the messenger RNA (mRNA) expression for the 23 known genes of matrix metalloproteinases (MMPs), 19 genes of ADAMTS, 4 genes of tissue inhibitors of metalloproteinases (TIMPs), and ADAM genes 8, 10, 12, and 17 in normal, painful, and ruptured Achilles tendons., Methods: Tendon samples were obtained from cadavers or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. Total RNA was extracted and mRNA expression was analyzed by quantitative real-time reverse transcription-polymerase chain reaction, normalized to 18S ribosomal RNA., Results: In comparing expression of all genes, the normal, painful, and ruptured Achilles tendon groups each had a distinct mRNA expression signature. Three mRNA were not detected and 14 showed no significant difference in expression levels between the groups. Statistically significant (P < 0.05) differences in mRNA expression, when adjusted for age, included lower levels of MMPs 3 and 10 and TIMP-3 and higher levels of ADAM-12 and MMP-23 in painful compared with normal tendons, and lower levels of MMPs 3 and 7 and TIMPs 2, 3, and 4 and higher levels of ADAMs 8 and 12, MMPs 1, 9, 19, and 25, and TIMP-1 in ruptured compared with normal tendons., Conclusion: The distinct mRNA profile of each tendon group suggests differences in extracellular proteolytic activity, which would affect the production and remodeling of the tendon extracellular matrix. Some proteolytic activities are implicated in the maintenance of normal tendon, while chronically painful tendons and ruptured tendons are shown to be distinct groups. These data will provide a foundation for further study of the role and activity of many of these enzymes that underlie the pathologic processes in the tendon.

We recently undertook expression profiling of all 19 human ADAMTS metalloproteinases (a disintegrin and metalloproteinase with thrombospondin motifs) in malignant and non-neoplastic breast tissue and showed that 11 of the ADAMTS genes are dysregulated in breast carcinoma. We identified a subgroup of ADAMTSs, based on functional and amino acid sequence similarity (ADAMTS1, 4, 5, 8 and 15), to be the focus of further study in breast carcinoma. Further RNA expression analysis by real-time PCR on a different cohort of 229 patients with breast cancer has identified ADAMTS8 as a predictor of poor overall survival (OS) (hazard ratio (HR) = 2.20, 95% C.I. = 1.29-3.74, p = 0.004) and confirmed ADAMTS15 as a predictor of prolonged relapse-free survival (RFS) (HR = 0.54, 95% C.I. = 0.32-0.89, p = 0.016). We explored the differences in survival of the 4 groups that could be categorized based on the expression levels of ADAMTS8 and ADAMTS15. For both RFS and OS, the group with high ADAMTS8 and low ADAMTS15 expression had a particularly poor prognosis. This group had a 3-fold higher chance of recurrence (HR = 3.03, 95% C.I. = 1.49-6.15, p = 0.001) and a greater than 5-fold higher chance of death (HR = 5.40, 95% C.I. = 2.16-13.5, p < 0.001) than the most favorable prognostic group. This prediction of poor prognosis by ADAMTS8 and ADAMTS15 expression was found to be independent of other classical clinicopathological factors. Results observed in FVB-PyMT mice, a robust transgenic model of highly metastatic breast carcinoma, fitted the expectation that relatively high expression levels of ADAMTS8 together with low expression levels of ADAMTS15 seen in human breast carcinoma are associated with a poor clinical outcome. In summary, ADAMTS8 and ADAMTS15 have emerged as novel predictors of survival in patients with breast carcinoma., ((c) 2005 Wiley-Liss, Inc.)

Rationale: Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology., Methods: We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis-infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry., Results: MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis-associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guérin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis-infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells., Conclusions: M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2-dependent MMP-1 secretion.

Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3(-/-) mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNFalpha converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-alpha (TNFalpha) processing. In addition, TNFalpha production increased in timp-3(-/-)-AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3(-/-)-AB myocardium. Timp-3(-/-)/tnfalpha(-/-) mice were generated and subjected to AB for comparative analyses with timp-3(-/-)-AB mice. This revealed the critical role of TNFalpha in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNFalpha, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNFalpha, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3(-/-)-AB mice. Notably, combining TNFalpha ablation with MMP inhibition completely rescued heart disease in timp-3(-/-)-AB mice. This study provides a basis for anti-TNFalpha and MMP inhibitor combination therapy in heart disease.

Angiogenesis is the process by which new blood vessels are formed from preexisting vasculature. It is an essential feature of the female reproductive cycle, embryonic development and wound repair. Angiogenesis has also been identified as a causal or contributing factor in several pathologies, including cancer, where it is a rate-limiting step during tumor progression. Matrix metalloproteinases (MMPs) are a family of soluble and membrane-anchored proteolytic enzymes that can degrade components of the extracellular matrix (ECM) as well as a growing number of modulators of cell function. Several of the MMPs, in particular the gelatinases and membrane-type 1 MMP (MT1-MMP), have been linked to angiogenesis. Potential roles for these proteases during the angiogenic process include degradation of the basement membrane and perivascular ECM components, unmasking of cryptic biologically relevant sites in ECM components, modulation of angiogenic factors and production of endogenous angiogenic inhibitors. This review brings together what is currently known about the functions of the MMPs and the closely related ADAM (a disintegrin and metalloproteinase domain) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families in angiogenesis and considers how this information might be useful in manipulation of the angiogenic process, with a view to constraining tumor progression., (Copyright 2005 Wiley-Liss, Inc.)

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of mRNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study the possible interactions between cancer and stromal cells during the development of breast cancer, and the results suggest that stromal cells are involved in carcinogenesis and tumor progression, which may have important implications for the biology and therapy of cancer.

Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecan-degrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.

Objective: To profile the expression of all known members of the matrix metalloproteinase (MMP), ADAMTS, and tissue inhibitor of metalloproteinases (TIMP) gene families in normal cartilage and cartilage from patients with osteoarthritis (OA)., Methods: Human cartilage was obtained from femoral heads at joint replacement for OA or following fracture to the femoral neck. Total RNA was purified, and gene expression was assayed using quantitative real-time polymerase chain reaction., Results: Several members of the above gene families were regulated in OA. Genes that showed increased expression in OA were MMP13, MMP28, and ADAMTS16 (all at P < 0.001), MMP9, MMP16, ADAMTS2, and ADAMTS14 (all at P < 0.01), and MMP2, TIMP3, and ADAMTS12 (all at P < 0.05). Genes with decreased expression in OA were MMP1, MMP3, and ADAMTS1 (all at P < 0.001), MMP10, TIMP1, and ADAMTS9 (all at P < 0.01), and TIMP4, ADAMTS5, and ADAMTS15 (all at P < 0.05). Correlation analysis revealed that groups of genes across the gene families were coexpressed in cartilage., Conclusion: This is the first comprehensive expression profile of all known MMP, ADAMTS, and TIMP genes in cartilage. Elucidation of patterns of expression provides a foundation with which to understand mechanisms of gene regulation in OA and potentially to refine the specificity of antiproteolytic therapies.

Matrix metalloproteinases (MMPs) are implicated in multiple sclerosis where one of their roles may be to facilitate the transmigration of circulating leukocytes into the CNS. Studies have focused on only a few MMPs, and much remains unknown of which of the 23 MMP family members is/are critical to the multiple sclerosis disease process. Using quantitative real time polymerase chain reactions, we have systematically analysed the expression of all 23 MMP members in subsets of leukocytes isolated from the blood of normal individuals. We found a distinctive pattern of MMP expression in different cellular populations: MMP-11, MMP-26 and MMP-27 were enriched in B cells, while MMP-15, MMP-16, MMP-24 and MMP-28 were prominent in T lymphocytes. Of interest is the enrichment of a majority of MMP members in monocytes: MMP-1, MMP-3, MMP-9, MMP-10, MMP-14, MMP-19 and MMP-25. MMP-2 and MMP-17 were also significantly represented in monocytes, although B cells had significant amounts of these MMPs. In correspondence with their strong expression of many MMP members, monocytes migrated more rapidly across a model of the blood-brain barrier in culture than T or B lymphocytes. Finally, we found higher levels of two of the monocyte-expressed MMPs in multiple sclerosis patients compared with normal individuals: MMP-2 and MMP-14. Tissue inhibitor of metalloproteinases (TIMP)-2 was also elevated in monocytes from multiple sclerosis patients, providing a mechanism for the reported activation of MMP-2 by MMP-14 and TIMP-2. These results emphasize that monocytes are prominent contributors of the neuroinflammation in multiple sclerosis through a mechanism that involves their high MMP expression and that they identify specific MMP members as targets for novel therapeutics in the disease.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.

Matrix metalloproteinases (MMPs) have long been thought of as critical factors regulating matrix degradation associated with cell invasion into ectopic tissue compartments during primary tumor growth and metastasis. One member of the MMP family historically linked to these invasive processes is MMP-9/gelatinase B. By studying a transgenic mouse model of de novo epithelial carcinogenesis, new roles for MMP-9 have emerged that broaden the view of its functional contribution to malignant progression. The combined implication of these studies suggest that MMP-9 functionally contributes to cancer development; however, its major regulatory role may be in its ability to activate poorly diffusible and/or matrix-sequestered growth factors that regulate epithelial and/or endothelial cell growth as opposed to regulating cellular invasion across basement membranes.

The recognition that matrix metalloproteinases (MMPs) facilitate tumor cell invasion and metastasis has led to the development of synthetic MMP inhibitors (MMPIs) as cancer therapeutic agents. Because several Phase III trials failed recently to show efficacy of broad-spectrum MMPIs in advanced cancer, the feasibility of MMPs as therapeutic targets has been challenged. However, it has not yet been determined whether MMPIs that have increased specificity may have greater benefit. We show that MMP-9 expression closely correlates with the progression of liver metastasis in a T-cell lymphoma model. MMPIs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data demonstrate a link between increased specificity of MMPIs and enhanced anticancer activity.

Replicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases (MMPs); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA. Articular cartilage was obtained from OA patients undergoing arthroplasty, with 'normal' cartilage from trauma surgery for hip fracture. Senescent cells were identified using the senescence-associated beta-galactosidase (SA-beta-gal) marker. Telomere length was assessed using Southern blot. MMP expression was measured at the mRNA level using Taqman RT-PCR. No SA-beta-gal staining was observed in control cartilage regardless of patient age. In contrast, SA-beta-gal staining was observed in damaged OA cartilage adjacent to the lesion. Cultured chondrocytes isolated from sites near a lesion contained a greater percentage of SA-beta-gal positive cells than cultures isolated from distal sites or normal cartilage. Mean telomere length was shorter in cells near the lesion compared to distal sites in the same joint; thus the former population has undergone cell division. The expression of collagenases MMP-1, -8 and -13 and tissue inhibitor of metalloproteinases (TIMP)-1 was altered in OA cartilage, but no difference was detected between lesion and distal sites in the same joint (i.e. no correlation was found between senescent cells and proteinase/ inhibitor expression).

Progressive renal disease as a result of renal fibrosis is caused in part by an impairment of the proteolytic machinery that normally regulates matrix turnover. The goal of the present study was to determine whether genetic deficiency of tissue inhibitor of metalloproteinases-1 (TIMP-1) could attenuate interstitial fibrosis caused by unilateral ureteral obstruction (UUO). Groups of wild-type (Timp-1) mice and TIMP-1-deficient (timp-1) mice were killed after 3 and 14 d of UUO or sham operation. Timp-1 mRNA levels were significantly increased 37- and 19-fold in the wild-type mice 3 and 14 d, respectively, after UUO operation. Matrix metalloproteinase-9 (MMP-9) activity fell in all UUO groups but remained significantly higher in the timp-1 group compared with the Timp-1 group. The degree of interstitial fibrosis (kidney collagen content and percentage of tubulointerstitial area stained with picrosirius red and collagen III) was significantly increased 14 d after UUO operation, but there was no difference between the Timp-1 and timp-1 groups. Many features of the fibrogenic response were similar between the Timp-1 and timp-1 groups, including the number of myofibroblasts and the induction of genes encoding procollagen III, fibronectin, and transforming growth factor-beta. After UUO operation, renal mRNA levels for Timp-3 and plasminogen activator inhibitor-1 were significantly higher in the TIMP-1-deficient mice. The results of this study show that elimination of TIMP-1 alone does not alter the severity of interstitial fibrosis. These findings may be due to compensation by other protease inhibitors such as TIMP-2, TIMP-3, and/or plasminogen activator inhibitor-1 or to the possibility that inhibition of intrinsic MMP activity does not constitute a profibrogenic event in the kidney.