Anna E. Kossakowska

Altered Balance Between Matrix Metalloproteinases and Their Inhibitors in Experimental Biliary Fibrosis

A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression and activities of collagen-degrading proteinases and their inhibitors during the progression of fibrosis. Expression of four members of the matrix metalloproteinase (MMP) family (MMP-2/gelatinase A, MMP-3, MMP-9/gelatinase B, and MMP-13) and three tissue inhibitors of metalloproteinases-1, -2, and -3 (TIMP-1, TIMP-2, and TIMP-3) were evaluated by Northern blot analysis of RNA from liver tissue isolated at 0, 2, 5, 10, 20, and 30 days after either a BDL or sham operation. In addition, we analyzed free gelatinase and TIMP activities by zymography and reverse zymography, respectively. We found that the proteolytic activities of MMP-2 and MMP-9 increased by 2 days after ligation, reached maximal levels at day 10, and remained high through the study period, whereas the gelatinolytic activities in plasma were unchanged. The increase in gelatinase activities was accompanied by an increase in the TIMP mRNA transcripts. TIMP-1 transcripts appeared at day 2, increased until day 10, and remained elevated throughout the study period. TIMP-2 and TIMP-3 transcripts become detectable on day 10 and remained stable afterwards. No corresponding increase in TIMP protein activity was detected by reverse zymography. This appears to result from the formation of TIMP/MMP complexes. These findings indicate a likely surplus in the BDL model of fibrosis of free gelatinases as compared with the TIMPs. Thus, excessive TIMP production is not a sufficient explanation for the observed extracellular matrix accumulation, but complex changes in the local MMP/TIMP balance may underlie the pathomechanisms of fibrosis.

Comparative analysis of the expression patterns of metalloproteinases and their inhibitors in breast neoplasia, sporadic colorectal neoplasia, pulmonary carcinomas and malignant non-Hodgkin's lymphomas in humans.

Matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play essential roles in the remodelling of the extracellular matrix (ECM). Results of in vivo and in vitro studies suggest that the balance between MMPs and TIMPs is altered in neoplasia, contributing to the invasive and metastatic properties of malignant tumours. In this study we have analysed the expression of five MMP genes and TIMP-1 and TIMP-2 in 37 benign and malignant lesions of human breast using Northern blot analysis. MMP-9 (92 kDa gelatinase) and MMP-11 (stromelysin 3) were most consistently expressed by carcinomas. Based on detection of either MMP-9 or MMP-11 mRNAs, we were able to distinguish between malignant and benign disease with a predictive accuracy of 90% with 94% sensitivity and 85% specificity. Subsequently, these results were compared with results for carcinomas of colon and lung and malignant non-Hodgkins lymphomas (NHL). Elevated MMP-9 and TIMP-1 expression was observed in all four systems. MMP-11 characterised all carcinomas as well as carcinomas in situ but was not detectable in NHL. Our data therefore argue that there are remarkably similar patterns of specific functions involved in ECM remodelling that correlate with malignancy in different human tumours of different histogenesis. However, MMP-11 expression is a characteristic of tumours of epithelial origin that is not found in lymphoid neoplasia. Thus it suggests that MMP-11 may play a regulatory role in the invasion and metastasis of carcinomas.

Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas.

Nine primary pulmonary carcinomas, one metastatic carcinoma, and two malignant pleural mesotheliomas have been analysed for the expression at the mRNA level of metalloproteinases (MPs) and tissue inhibitors of MPs (TIMPs). In situ hybridisation showed TIMP-1 and TIMP-2 transcripts predominantly over tumour stroma and gelatinases evenly distributed over both stromal and tumour cells. While both TIMP-1 and TIMP-2 were expressed in non-neoplastic lungs (NNL) as well as in carcinomas, stromelysin 3 (ST3), 92 kDa gelatinase and interstitial collagenase were expressed only by carcinomas. Expression of these MPs by carcinomas was independent of histologic type and such tumour features as fibrosis or necrosis. The consistent expression of ST3 by all of the carcinomas examined and absence of its expression in NNL indicates that ST3 production is likely associated with the malignant phenotype. However, since 92 kDa gelatinase and interstitial collagenase transcripts were found in some but not all tumour samples, their expression is not a uniform feature of pulmonary carcinomas. The possible prognostic significance of the expression of the latter two enzymes by carcinomas remains to be established.

Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: Comparison with normal bone marrow cells

We compared the expression of matrix metalloproteinases (MMP‐2 and MMP‐9) and tissue inhibitors of metalloproteinases (TIMP‐1 and TIMP‐2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL‐60, K‐562 and KG‐1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP‐9 and/or MMP‐2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP‐1 and TIMP‐2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP‐1, the HL‐60 cells also expressed TIMP‐2. In contrast, normal steady‐state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP‐2 or MMP‐9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP‐9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP‐1 and TIMP‐2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP‐2, the activated form of this enzyme was found in media conditioned by cells obtained from long‐term cultures of normal and AML bone marrow adherent layers. Our finding of up‐regulated production of gelatinases, TIMP‐1 and TIMP‐2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.

Expression Pattern of Metalloproteinases and Their Inhibitors Changes with the Progression of Human Sporadic Colorectal Neoplasia

Several studies have implicated the extracellular matrix-degrading metalloproteinases (MMPs) as essential agents in tumor cell invasion and metastasis. In the present study, we have investigated the patterns of expression of a number of MMPs and their specific tissue inhibitors (TIMP-1 and TIMP-2) in human colonic tissue samples that represent various stages of progression from adenomas showing different degrees of dysplasia to adenocarcinomas. We assessed levels of mRNA by Northern blot analysis and the results were measured semiquantitatively by densitometry. In total, we analyzed nine adenomas of varying size and with varying degrees of dysplasia. three adenomas with adenocarcinoma (malignant polyps). and five adenocarcinomas. Although expression of MMP and TIMP mRNA was highly intercorrelated, transcripts for stromelysin 3 and TIMP-2 (high) showed the strongest relation to the neoplastic process. Detection of stromelysin 3 mRNA accompanied a diagnosis of severe dysplasia or malignancy, whereas levels of TIMP-2 (high) mRNA transcripts permitted finer distinctions on the neoplastic continuum. These data indicate changes within extracellular matrix acquired during the process of malignant transformation of human sporadic colorectal neoplasia.

Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.

Matrix metalloproteinases and their tissue inhibitors - expression, role and regulation in human malignant non-Hodgkin's lymphomas.

Human malignant non-Hodgkins lymphomas (NHL) represent a heterogeneous group of neoplasms, which vary in their clinical behavior and pathophysiology. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been shown to play a role in the pathophysiology and clinical aggressiveness of human NHL. In this setting, MMP-9 and TIMP-I appear to be the most important members of the MMP and TIMP families, and overexpression of both correlates with a poor clinical outcome of patients with NHL. MMP-9 and TIMP-I, however, act through different mechanisms and are produced by different cell types. Expression of both is upregulated by interleukin-6 (IL-6), a cytokine that is known as one of the factors involved in the pathophysiology of human NHL. In this review we summarize the complex regulation of MMP and TIMP expression in human NHL and propose a mechanism by which MMP-9, TIMP-I and IL-6 may influence the biology of these tumors.

Elevated plasma gelatinase A (MMP-2) activity is associated with quiescent Crohn's Disease

Crohn’s disease (CD) is an idiopathic inflammatory condition characterized by an unpredictable clinical course and frequent relapses. Stenosis, fissures, and fistulae, seen in 20–40% of patients, likely result from abnormal extracellular matrix (ECM) metabolism. Such complications may necessitate an alteration of medical therapy or surgical intervention. Many assays for markers of CD relapse have been investigated, but at present no test is implemented clinically.

Immunoglobulin and T-Cell Receptor Gene Rearrangements in Lesions of Mucosa-Associated Lymphoid Tissue

Twenty-one cases of mucosa-associated lymphoid tissue (MALT) lesions have been analyzed by Southern blot for immunoglobulin heavy chain (IgH) and T-cell receptor β chain (TcRβ) gene rearrangements (GR). The sites included colon, stomach, liver, nasopharynx, salivary and lacrimal gland, conjunctiva, tonsil, breast, and lung. Two of the lesions (parotid and conjunctiva) were malignant lymphomas and one case showed lymphoproliferative disorder. In the cases of malignant lymphomas, IgH GR were detected, and in the case of lymphoproliferative disorder, both IgH and TcRβ genes were rearranged. Among the remaining 18 cases, 9 showed inflammatory infiltrate, 3 lymphoid hyperplasia, 3 atypical lymphoid hyperplasia, 1 carcinoma of the tonsil, 1 breast carcinoma, and one was a sample of normal Peyer s patches. Among these 18 cases, 3 showed TcRβ GR, 6 showed double IgH and TcRβ GR. and 4 IgHβgH GR. Often multiple rearranged bands were observed, composing 10–30% of the total DNA analyzed. The control tissue (Peyers patches) showed no GR. Because IgH and TcRβ GR are used to determine monoclonal proliferations of T and B lymphocytes, which occur in malignant lymphomas, it is vital to determine the specificity of such a test. This report stresses the fact that in MALT lesions false-positive results are not uncommon and therefore the results of IgH and TcRβ GR studies have to be interpreted with caution. The presence of multiple GR in the inflammatory lesions indicates proliferation of minor monoclonal populations that can be detected with the use of Southern blot technology.

Leukocyte elastase in murine and human non-Hodgkin lymphomas.

Extracellular proteases play a crucial role in the invasive behavior of normal and transformed leukocytes. Thus far, however, most of the attention has been focused on members of the family of matrix metalloproteinases. In this work, we show that lymphoma cells can express leukocyte elastase (LE) and recruit the enzyme at their surface via ICAM‐1. The expression of LE by lymphoma cells was augmented significantly by stimulation with IL‐6 and IL‐13, both of which also induced the expression of MMP‐9. Although LE and IL‐13 transcripts were detected in several non‐Hodgkin’s lymphomas, immunohistochemical analysis of lymphoma tissues also showed that LE was strongly expressed in infiltrating leukocytes. Given the spectrum of key molecules that can be cleaved by LE and that LE and MMP‐9 are involved in the invasive behavior of normal or transformed leukocytes, our results raise the hypothesis that LE plays a crucial role in the multistep processes of inflammation and lymphoma metastasis.