Involvement of specific matrix metalloproteinases during tumor necrosis factor/IFNgamma-based cancer therapy in mice

The potent antitumor activity of tumor necrosis factor (TNF) in combination with IFN-gamma can only be applied in local regimens due to their strong proinflammatory properties. It has been shown that the broad-spectrum matrix metalloproteinase (MMP) inhibitor BB-94 protects against TNF/IFNgamma-induced toxicity without blocking the antitumor effect. Here, we tried to explain this protective role of BB-94 and sought to assign roles to specific MMPs in TNF/IFNgamma-induced toxicity. By studying the expression of MMP genes in different organs and in the tumor, we observed that the expression levels of MMP-7, MMP-8, MMP-9, and MMP-12 and tissue inhibitor of metalloproteinase-4 are clearly up-regulated in the liver during therapy. MMP-8 and MMP-9 are also up-regulated in the lung and kidney, respectively. In the tumor, most MMP genes are expressed, but only MMP-3 is up-regulated during TNF/IFNgamma treatment. Using MMP-deficient or double-deficient mice, we have shown a mediating role for MMP-3 during TNF/IFNgamma treatment in tumor-free and B16BL6 melanoma-bearing mice. By contrast, MMP-12 seemed to have some protective role in both models. However, because most phenotypes were not extremely outspoken, we have to conclude, based on the set of MMP-deficient mice we have studied, that inhibition of a single MMP will probably not increase the therapeutic value of TNF/IFNgamma, but that rather, broad-spectrum MMP inhibitors will be required.     

Matrix metalloproteinases and their inhibitors: promising novel biomarkers in severe sepsis?

The multicenter study conducted by Lorente and coworkers published in the previous issue of Critical Care demonstrates that matrix metalloproteinase (MMP)-9 and MMP-10 and their inhibitor tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are promising novel biomarkers to predict severity and outcome of sepsis. In recent years MMPs have emerged as biomarkers in a variety of diseases, such as sepsis, coronary artery disease, cancer, heart failure, chronic lung disease and rheumatoid arthritis. MMPs constitute a family of proteinases that are expressed during developmental, physiological, and pathophysiological processes, for example as a response to infection. Excessive inflammation following infection may cause tissue damage, and MMPs are implicated in causing this immunopathology. The activity of MMPs is regulated by secretion of specific inhibitors (TIMPs). Studies using MMP inhibitors and MMP knockout mice indicate that MMPs play an essential role in infection and in the host response to infection. The measurement of MMP-9 and MMP-10 and their inhibitor TIMP-1 in the intensive care setting could be an attractive noninvasive tool for determination of outcome of septic patients.     

MMP-1 drives immunopathology in human tuberculosis and transgenic mice

Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis-driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.     

Evaluation of metalloproteinases 2 and 9 and their inhibitors in physiologic and pre‐eclamptic pregnancy

Matrix metalloproteinases (MMPs) are a family of zinc and calcium‐dependent endopeptidases involved in remodeling and physiological homeostasis of extracellular matrix (ECM). The metalloproteinases activity is predominantly modulated by specific tissue inhibitors of matrix metalloproteinases (TIMPs). The balance between MMPs and TIMPs is likely to play an important role in remodeling uterine arteries in pregnancy, and it may represent means by which vasodilatation is maintained in later pregnancy. Moreover, increased levels of MMPs and in particular MMP‐2 play a role in the vascular alterations induced by hypertension. The aim of this study was the evaluation of MMP‐2 and ‐9, along with their inhibitors TIMP‐1 and ‐2, in pre‐eclamptic women compared with normotensive pregnancy and non‐pregnant women. Fourteen pre‐eclamptic women were compared with 37 normotensive women in different gestational age and 21 non‐pregnant women. Multiplexed sandwich enzyme‐linked immunosorbent assay was used to measure MMPs and TIMPs simultaneously. MMP‐2 levels were significantly higher in pre‐eclamptic women vs. both non‐pregnant and physiologic pregnant women. MMP‐9 concentrations were significantly higher in physiologic pregnant vs. non‐pregnant women. The serum levels of TIMP‐1 were significantly higher in pre‐eclamptic vs. both non‐pregnant and physiologic pregnant women. TIMP‐2 values were higher in physiologic pregnant women and pre‐eclamptic women vs. non‐pregnant women. A positive correlation between MMP‐9 values and gestational age was observed in normal pregnant women. Results of the present investigation confirm that MMP‐2 and TIMP‐1 values are significantly higher in preeclampsia. We confirm that the modification of the fine balance between MMPs and their inhibitors plays a greater role in the structural and functional vascular changes of women with complicated pregnancies. J. Clin. Lab. Anal. 23:88–92, 2009. © 2009 Wiley‐Liss, Inc.     

Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction

Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.     

Circulating matrix metalloproteinases and their inhibitors in hypertension

Growing experimental evidence indicates that matrix metalloproteinases (MMPs) are implicated in many cardiovascular diseases including hypertension and its chronic complications. It is now clear that MMPs have many more substrates other than components of the extracellular matrix. In fact, intracellular targets now include those associated with the cardiovascular system. Clinical studies have suggested that circulating MMPs may predict cardiovascular morbidity and mortality. It is highly probable that increased MMPs may predispose hypertensive patients to additional complications and clinical sequelae. In this article, we review the basic principles linking MMP activity with hypertension and summarize clinical studies examining two specific MMPs (MMP-2 and -9) in hypertension. We also discuss how antihypertensive drugs may affect MMPs and their endogenous inhibitors, i.e., tissue inhibitors of matrix metalloproteinases (TIMPs). Circulating MMPs may predict increased risk of developing cardiovascular complications associated with hypertension. As such, patients could benefit from early pharmacologic intervention including use of MMP inhibitors.     

Matrix metalloproteinases: influence on smooth muscle cells and atherosclerotic plaque stability

Atherosclerotic plaque rupture, with subsequent occlusive thrombosis, is the underlying cause of most cases of sudden cardiac death. Matrix metalloproteinases (MMPs) are thought to mediate the progression of stable atherosclerotic lesions to an unstable phenotype that is prone to rupture through the destruction of strength-giving extracellular matrix (ECM) proteins. Smooth muscle cells secrete and deposit ECM proteins and are, therefore, considered protective against atherosclerotic plaque destabilization. However, similar to inflammatory cells (e.g., macrophages), smooth muscle cells release numerous MMPs that are capable of digesting ECM proteins. Thus, the interaction of smooth muscle cells and MMPs in atherosclerotic plaques is complex and not fully understood. Recently, research into the roles of MMPs and their endogenous inhibitors (tissue inhibitors of metalloproteinases), and their effects on smooth muscle behavior during plaque destabilization has been aided by the development of reproducible animal models of plaque instability. A plethora of studies has demonstrated that MMPs directly modulate smooth muscle behavior with both beneficial and deleterious effects on atherosclerotic plaque stability, in addition to their canonical effects on ECM remodeling. Consequently, broad-spectrum MMP inhibition may inhibit plaque-stabilizing mechanisms, such as smooth muscle cell growth, while conversely retarding ECM destruction and subsequent rupture. Hence the development of selective MMP inhibitors, that spare inhibitory effects on smooth muscle cell function, may be useful therapies to prevent plaque rupture and in this regard MMP-12 appears to be a particularly attractive target.     

Protease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiation

We previously showed that human and murine 3T3-F442A preadipocytes produced and released matrix metalloproteinases (MMPs) 2 and 9 and that a treatment by MMP inhibitors resulted in the blockade of murine fat cell adipose conversion. In parallel, investigators reported that other protease inhibitors, the human immunodeficiency virus (HIV) protease inhibitors (PIs) involved in lipodystrophy in humans, also reduced the adipocyte differentiation process of several murine cell lines. The present work was performed to define the effects of MMP inhibitors and HIV-PIs on the human adipocyte differentiation process, to clarify the involvement of MMPs in the control of human adipogenesis, and to determine whether HIV-PIs interact with MMPs in the control of this process. The effect of two MMP inhibitor and four HIV-PI treatments on the differentiation of primary culture human preadipocytes, as well as the putative relationships between HIV-PIs and MMP-2 and -9 expression, release, or activity were investigated. We showed that MMP inhibitors and HIV-PIs reduced the human adipocyte differentiation process as assessed by the decrease of cell protein and/or triglyceride contents and expression of fatty acid binding protein and hormone-sensitive lipase, two adipocyte markers. Unlike MMP inhibitors, HIV-PIs were devoid of any effect per se on recombinant MMP-2 and 9 activities but reduced the expression and release of MMP-9 by human preadipocytes. Thus, the present study indicates that the modulation of the extracellular matrix components through the production and/or activity of MMPs, and, more precisely, MMP-9 might be a key factor in the regulation of human adipose tissue development.     

Matrix metalloprotease-2 and -9 concentration and activity in serum and culture medium samples: a methodological reappraisal.

BACKGROUND: Matrix metalloproteases (MMPs) play an important role in cardiovascular remodeling by degrading the extracellular matrix. The aim of this study was to compare two different methods for MMP-2 and MMP-9 concentration and activity determination. METHODS: MMP-2 and -9 levels were measured by immunometric and enzymatic assays to determine total and active levels. The two procedures differ in assay principle and in the extent of cross-reactions with interfering substances present in biological samples. Both human serum and culture medium from an ex vivo human model of intimal hyperplasia were checked. RESULTS: All methods were able to detect MMP-2 and -9 with similar levels of sensitivity, reproducibility and accuracy, and furnished positively related results, although significantly different, in both types of sample. Both systems were able to detect changes in MMP production such as the time-course of MMP-2 and -9 release by cultured saphenous vein associated with intima hyperplasia progression. CONCLUSIONS: Our data indicate that different values for MMP concentrations can be obtained using different analytical methods, even if they are intrinsically reliable. This suggests that methodological differences should be taken into account when comparing MMP results from different studies.     

Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms

Abdominal aortic aneurysms represent a life-threatening condition characterized by chronic inflammation, destructive remodeling of the extracellular matrix, and increased local expression of matrix metalloproteinases (MMPs). Both 92-kD gelatinase (MMP-9) and macrophage elastase (MMP-12) have been implicated in this disease, but it is not known if either is necessary in aneurysmal degeneration. We show here that transient elastase perfusion of the mouse aorta results in delayed aneurysm development that is temporally associated with transmural mononuclear inflammation, increased local production of several elastolytic MMPs, and progressive destruction of the elastic lamellae. Elastase-induced aneurysmal degeneration was suppressed by treatment with a nonselective MMP inhibitor (doxycycline) and by targeted gene disruption of MMP-9, but not by isolated deficiency of MMP-12. Bone marrow transplantation from wild-type mice prevented the aneurysm-resistant phenotype in MMP-9-deficient animals, and wild-type mice acquired aneurysm resistance after transplantation from MMP-9-deficient donors. These results demonstrate that inflammatory cell expression of MMP-9 plays a critical role in an experimental model of aortic aneurysm disease, suggesting that therapeutic strategies targeting MMP-9 may limit the growth of small abdominal aortic aneurysms.     

基质金属蛋白酶及其组织抑制因子在心肌梗死后心室重构中的作用

心肌梗死后心室重构是心力衰竭产生的重要原因,其中细胞外基质的改建是重要的分子机制。基质金属蛋白酶(MMPs)是降解细胞外基质的重要蛋白水解系统。基质金属蛋白酶组织抑制因子(TIMPs)是MMPs的内源性抑制系统。MMPs/TIMPs平衡失调是心肌梗死后胶原网络重构的重要调节因素之一。现就MMPs和TIMPs在心肌梗死后心室重构中的作用及基质金属蛋白酶抑制剂抗重构治疗的研究进展作一综述。     

Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis

Degradation of ECM, particularly interstitial collagen, promotes plaque instability, rendering atheroma prone to rupture. Previous studies implicated matrix metalloproteinases (MMPs) in these processes, suggesting that dysregulated MMP activity, probably due to imbalance with endogenous inhibitors, promotes complications of atherosclerosis. We report here that the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) can function as an MMP inhibitor. TFPI-2 diminished the ability of the interstitial collagenases MMP-1 and MMP-13 to degrade triple-helical collagen, the primary load-bearing molecule of the ECM within human atheroma. In addition, TFPI-2 also reduced the activity of the gelatinases MMP-2 and MMP-9. In contrast to the "classical" tissue inhibitors of MMPs (TIMPs), TFPI-2 expression in situ correlated inversely with MMP levels in human atheroma. TFPI-2 colocalized primarily with smooth muscle cells in the normal media as well as the plaque's fibrous cap. Conversely, the macrophage-enriched shoulder region, the prototypical site of matrix degradation and plaque rupture, stained only weakly for TFPI-2 but intensely for gelatinases and interstitial collagenases. Evidently, human mononuclear phagocytes, an abundant source of MMPs within human atheroma, lost their ability to express this inhibitor during differentiation in vitro. These findings establish a new, anti-inflammatory function of TFPI-2 of potential pathophysiological significance for human diseases, including atherosclerosis.     

Relationship between matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases systems and autoantibody patterns in systemic sclerosis

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis that reflects an imbalance between collagen production and degradation. Matrix metalloproteinases (MMPs) are a family of endopeptidases involved in the remodelling of extracellular matrix (ECM). This activity is controlled by tissue inhibitors of MMP (TIMPs). Aim of this study was the evaluation of MMP-9/TIMP-1 and MMP-2/TIMP-2 systems in patients with SSc. DESIGN AND METHODS: SearchLight Human MMP Array 1 was used to measure MMPs and TIMPs in 32 SSc patients and 32 matched healthy controls. RESULTS: SSc patients showed higher values of both MMP-9 and TIMP-1 in comparison with controls. The patients with anticentromere antibodies (ACA) positivity showed higher values of MMPs and TIMPs in comparison with either controls or the patients with anti-Scl70-positive antibodies. CONCLUSION: Results of this investigation suggest that SSc patients with ACA positivity, after a primary fibrogenetic noxa, react with a more abundant release of MMP/TIMP, whereas patients with anti-Scl70 antibody show a normal response.     

Cell-cell contact activation of fibroblasts increases the expression of matrix metalloproteinases

BACKGROUND: We recently found that direct homotypic cell-cell contacts between human dermal fibroblasts induce a novel form of cell activation leading to non-apoptotic programmed cell death. As the major features of this process we identified massive induction of cyclo-oxygenase-2 and production of inflammatory prostaglandins. On the surface of the decomposing spheroids, activation of the major extracellular proteolytic cascade, plasminogen activation, associated with surface exposure of alpha-enolase, took place. AIM: To further characterize pericellular proteolysis by cell-cell contact-activated fibroblasts we studied the role of the other major extracellular proteolytic system, matrix metalloproteinases (MMPs). METHODS: MMP expression in fibroblast clusters and monolayers was compared using mRNA microarrays and immunoblot analyses. The activities of MMPs were confirmed using MMP inhibitors and caseinolysis. RESULTS: In microarrays MMP-1, -10, and -14 (MT1-MMP) were induced 5.8-, 106-, and 5.6-fold, respectively. These findings were confirmed by immunoblotting. Radial caseinolysis showed low level of proteolytic activity in spheroid-conditioned media; ilomastat, a general inhibitor of MMPs, suppressed 50% of the proteolytic activity thus confirming it to be at least in part due to MMPs. A cocktail of tetracycline-derived MMP inhibitors suppressed lactate dehydrogenase (LDH) release only 11%, and if combined with aprotinin 28%. CONCLUSIONS: Cell-cell contact activation of fibroblasts induced MMP-1, -10, and MT1-MMP expression, suggesting similar signaling to that in inflammation and cancer.     

Physiological matrix metalloproteinase concentrations in serum during childhood and adolescence, using Luminex Multiplex technology.

Matrix metalloproteinases are a family of zinc-dependent proteinases which are involved in the breakdown and remodeling of extracellular matrix. As children grow and adolescents reach pubescence, their bodies undergo changes that require age-related morphogenesis of the extracellular matrix, possibly requiring unique patterns of matrix metalloproteinase (MMP) expression during periods of rapid tissue growth (i.e., childhood) or accelerated tissue remodeling and expansion (i.e., adolescence). Therefore, we have characterized age-specific and gender-specific differences in circulating concentrations of MMPs (specifically MMP-1, -2, -3, -8 and -9) in 189 serum samples obtained from healthy subjects, aged 2-18 years. MMP concentrations were measured using Fluorokine MultiAnalyte Profiling kits and a Luminex Bioanalyzer, as well as by commercial ELISA. Serum levels of MMP-1, -2, -3, -8, and -9 in healthy pediatric subjects represent log-normal distributions. MMP-2 was significantly negatively correlated with age (r=-0.29; p<0.001), while MMP-3 was significantly positively correlated with age (r=0.38; p<0.001). Although plasma, not serum, is considered the appropriate blood sample for measurement of MMP-8 and -9, serum levels of MMP-8 and -9 were also found to be highly positively correlated with each other (r=0.76; p<0.01). MMP results obtained by Fluorokin MultiAnalyte Profiling methods correlated well with conventional ELISA methods and use of this technology provided several advantages over ELISA.     

EMMPRIN、MMP-2和MMP-9在恶性淋巴瘤中的表达及其临床意义

目的 探讨恶性淋巴瘤中细胞外基质金属蛋白酶诱导因子（EMPRIN）的表达及其与基质金属蛋白酶（MMP,）表达和疾病进展的关系。方法 运用实时定量PCR法检测了81例恶性淋巴瘤患者淋巴瘤组织中EMMPRIN、MMP-2和MMP-9的表达。同时通过电镜对淋巴瘤组织切片中肿瘤浸润血管情况进行观察。结果 与淋巴结反应性增生（8例）相比，不同类型的恶性淋巴瘤均高表达EMMPRIN、MMP-2和MMP-9。EMMPRIN来自淋巴瘤细胞。Ann Arbor分期为Ⅲ-Ⅳ、伴多个淋巴结外病变和国际预后指数分组为高危组的患者上述基因的表达水平显著升高。此外，EMM—PRIN、MMP-2和MMP-9高表达者可见淋巴瘤细胞浸润血管。结论 EMMPRIN表达可促进恶性淋巴瘤MMPS的表达，并与疾病进展密切相关。     

Proteolyzed matrix as a template for the regulation of tumor progression.

Pericellular proteolysis plays a pivotal function in cell invasion, a hallmark of tumor growth and metastasis. The minidegradome constituted of two matrix metalloproteinases (MMP), i.e. MMP-2 and MT1-MMP, associated with tissue inhibitor of metalloprotease-2 (TIMP-2) and integrin (alpha(v)beta(3)) or CD(44), is mainly involved in such invasive program. It catalyzes matrix degradation but, alternatively, proteolytic exposure of matricryptic sites or matrikines liberation by those enzymes regulates either positively or negatively tumor cell migration. That applies to types I and IV collagens, elastin, laminin 5, as described here, but such phenomenon might be extended to other matrix macromolecules. The development of tumors from epithelium origin is related to aging. Senescent fibroblasts are characterized by increased expression of MMPs, (particularly collagenase-1 (MMP-1) and stromelysin-1 (MMP-3)) and deposited matrix by those aged cells was shown to favor cancer cell growth. Thus, compositional variation of matrix-surrounding tumor cells, with formation of matricryptic sites and matrikines, can be considered as one main epigenetic factor contributing to tumor progression. A matrix-directed pharmacological approach in cancer is now emerging.