Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transformation-specific matrix metalloproteinases, MMP-7 and MMP-13, are present in epithelial cells of keratoacanthomas.

Keratoacanthomas are rapidly growing hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological factors. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -19 and p16 and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in 16/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in fibroblasts in both tumors. MMP-19 was upregulated in proliferating epithelium of keratoacanthomas as was p16. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-19 and p16 could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.     

Investigation of genetic alterations associated with the grade of astrocytic tumor by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a technique that allows the detection of losses and gains in DNA copy number across the entire genome. We used CGH to study the genetic alterations that occur in primary astrocytomas, including 14 glioblastomas (GBM), 12 anaplastic astrocytomas (AA), and 7 low-grade astrocytomas (LGA). The average numbers of total aberrations in GBM, AA, and LGA were 9.7, 5.4, and 4.0, respectively. The average number of DNA sequence losses in GBM was significantly higher than that in AA or LGA (P < 0.01). Frequently altered regions (> eight cases) observed in all grades of astrocytoma were 7p13-p12 (gain), 7q31 (gain), 8q24.1-q24.2 (gain), 9p21 (loss), 10p12-p11 (loss), 10q22-qter (loss), 13q21-q22 (loss), and 20q13.1-q13.2 (gain). Loss of 9p, 10p, or 10q, and the gain or amplification of 7p, were observed frequently in GBM (64%, 57%, 64%, and 50% of cases, respectively). Frequent alterations found in AA were losses of 9p, 10q, and 13q, and gains of 1q, chromosome 7, 11q, and Xq. Whereas 7p13-p11 amplification occurred exclusively in cases with the loss of all or part of chromosome 10, this change never occurred in cases having an increase in copy number of 8q, which was the most frequent change observed in LGA (four of seven cases). These results may indicate that an increase in copy number of 8q is an important event in GBM, with a genetic pathway, which is distinct from that in GBM with 7p amplification. Genes Chromosomes Cancer 21:340–346, 1998. © 1998 Wiley-Liss, Inc.     

Expression of MMP-10, MMP-21, MMP-26, and MMP-28 in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive cutaneous tumor with poor outcome and increasing incidence. We examined by immunohistochemistry the expression of three novel matrix metalloproteinases (MMPs)—MMP-21, MMP-26, and MMP-28—in 44 primary MCC tumors and six lymph node metastases while MMP-10 served as a positive control. Their mRNA expression was also studied in the UISO MCC cell line basally and after various stimulations using quantitative real-time PCR. MMP-28 was observed in tumor cells of 15/44 samples especially in tumors <2 cm in diameter (p = 0.015) while 21/44 specimens showed MMP-28 in the tumor stroma. Expression of MMP-21 was demonstrated in tumor cells of 13/43 samples. MMP-26, instead, was positive in stromal cells (17/44) and its expression associated with tumors ≥2 cm in diameter (p = 0.006). Stromal expression of MMP-10 was the most frequent finding of the studied samples (31/44), but MMP-10 was detected also in tumor cells (17/44). Most of the metastatic lymph nodes expressed MMP-10 and MMP-26. MMP-10, MMP-21, and MMP-28 mRNAs were basally expressed by the UISO cells, and the corresponding proteins were detectable by immunostaining of cultured cells. IFN-α and TNF-α downregulated MMP-21 and MMP-28 expression. Our results suggest that novel MMPs may have a role in MCC pathogenesis: especially that MMP-26 expression in stroma is associated with larger tumors with poor prognosis. Expression of MMP-21 and MMP-28 seems to associate with the tumors of lesser malignant potential. We also confirm the previous finding on the role of MMP-10 in MCC pathogenesis.     

Inverse relationships between the expression of MMP-7 and MMP-11 and predictors of poor prognosis of papillary thyroid carcinoma

AIMS: Matrix metalloproteinases (MMPs) have various functions that play roles in carcinoma development. In this study, we investigated the expression of two representative MMPs, MMP-7 and MMP-11, in papillary thyroid carcinomas. METHODS: We immunohistochemically investigated the expression of these MMPs in 196 cases of papillary carcinoma. RESULTS: A high level of MMP-7 expression was observed in 56 cases (28.3%). The expression level was significantly decreased in cases showing large tumour, N positivity, large pT and poor differentiation. MMP-11 expression was high in 119 cases (60.1%). The expression level was inversely related to tumour size, N factor, pT factor, pN factor, extrathyroid extension, and poor differentiation. CONCLUSIONS: In contrast to other MMPs, MMP-7 and MMP-11 are inversely linked to aggressive characteristics of papillary thyroid carcinoma and their down-regulations may even be a marker of poor prognosis in patients.     

In vitro Inhibition of Human MMP-2 Collagenolytic and Gelatinolytic Activity by Neutralizing Monoclonal Antibodies

Matrix metalloproteinases (MMPs) have been implicated in the degradation of the extracellular matrix in normal and pathological tissue remodelling. Among the MMPs, MMP-2 is the most commonly studied protease that has been involved in cancer, inflammation, infective diseases, degenerative diseases of the brain and vascular diseases. In this study, monoclonal antibodies (MAbs) were generated against human MMP-2, purified, characterized and tested for their ability to inhibit the enzymatic activity of MMP-2. Out of 12 positive clones generated against MMP-2, 2 clones (F2-1-11 and G8-25-5) were selected for further characterization. The selected clones react specifically with human pro and active form of MMP-2 in enzyme linked immunosorbant assay (ELISA), dot immunobinding assay (DIA) and Western blot and do not cross react with other human metalloproteinases or MMP-2 from other species. Additionally, these MAbs (F2-1-11 and G8-25-5) selectively inhibit collagenolytic and gelatinolytic activity of APMA ((p-aminophenylmercuric acetate)-activated-pro-MMP-2 and MMP-2, respectively.     

Expression and role of matrix metalloproteinases MMP-2 and MMP-9 in human spinal column tumors

Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immuno-histochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.© Kluwer Academic Publishers 1998     

Matrix metalloproteinase expression is related to angiogenesis and histologic grade in spindle cell soft tissue neoplasms of the extremities

We defined the immunocytochemical expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in benign soft tissue neoplasms, fibromatoses, and sarcomas, together with the activity of gelatinase MMPs and TIMPs measured by zymography and reverse zymography in a subset of cases. The most strongly expressed MMP in all tumors was MMP-1, with weaker expression of MMP-10, MMP-11, and MMP-14 in most tumors. Nuclear expression of MMP-1, MMP-8, and MMP-13 was an unusual feature. TIMP-2 was expressed in all tumors, with stronger expression in fibromatoses than in sarcomas. Fibromatoses and high-grade sarcomas showed greater MMP-1 expression than other groups, and endothelial MMP-2 expression was more extensive in sarcomas. Differences in MMP and TIMP expression might be linked to the biologic behavior of soft tissue neoplasms. The activation of endothelial MMP-2 linked to widespread MMP-14 expression provides a mechanism for sarcomas to modulate their matrix and facilitate angiogenesis.     

Gingival fibroblasts inhibit MMP-1 and MMP-3 activities in an ex-vivo artery model

The main arterial pathologies can be associated with a deregulation of remodeling involving matrix metalloproteinases (MMPs), whereas gingival healing is characterized by an absence of fibrosis or irreversible elastin/collagen degradation. The aim of our study was to evaluate the effect of gingival fibroblasts on MMP-1 and MMP-3 secretion in an organotypic artery culture. MMP-1 and MMP-3 secretions and activities (dot blots, zymography, ELISA) were evaluated in coculture of rabbit artery in the presence or not of gingival fibroblasts. MMP-1/TIMP-1 and MMP-3/TIMP-1 complexes forms were measured by ELISA. Complementary studies were performed using human aortic smooth muscle cells cocultured with adventitial, dermal, or gingival fibroblasts. Our results indicated that MMP-1 and MMP-3 free-forms activities were significantly reduced in coculture. This inhibition was linked to a significant increase of TIMP-1 leading to formation of TIMP-1/MMPs complexes. Due to the presence of gingival fibroblasts, the decrease in MMP-1 and MMP-3 efficiency thus contributes to diminish the degradation of artery. This cellular therapy strategy could be promising in artery pathologies treatment.     

Gingival Fibroblasts Inhibit MMP-1 and MMP-3 Activities in an Ex-Vivo Artery Model

The main arterial pathologies can be associated with a deregulation of remodeling involving matrix metalloproteinases (MMPs), whereas gingival healing is characterized by an absence of fibrosis or irreversible elastin/collagen degradation. The aim of our study was to evaluate the effect of gingival fibroblasts on MMP-1 and MMP-3 secretion in an organotypic artery culture. MMP-1 and MMP-3 secretions and activities (dot blots, zymography, ELISA) were evaluated in coculture of rabbit artery in the presence or not of gingival fibroblasts. MMP-1/TIMP-1 and MMP-3/TIMP-1 complexes forms were measured by ELISA. Complementary studies were performed using human aortic smooth muscle cells cocultured with adventitial, dermal, or gingival fibroblasts. Our results indicated that MMP-1 and MMP-3 free-forms activities were significantly reduced in coculture. This inhibition was linked to a significant increase of TIMP-1 leading to formation of TIMP-1/MMPs complexes. Due to the presence of gingival fibroblasts, the decrease in MMP-1 and MMP-3 efficiency thus contributes to diminish the degradation of artery. This cellular therapy strategy could be promising in artery pathologies treatment.     

Differential expression of stromal MMP-1, MMP-9 and TIMP-1 in basal cell carcinomas of immunosuppressed patients and controls

Matrix metalloproteinases (MMPs) have an important role in the initiation, growth, and invasion of malignant tumors. Basal cell cancer (BCC) is the most common human malignancy. The risk of BCC is 10–16 times higher among organ transplant recipients compared with the nontransplanted population. The aim of this study was to compare the expression of several MMPs and their tissue inhibitors (TIMPs) in BCCs from kidney transplant recipients and controls. Expression of MMPs-1, -7, -8, -9, -10, -13, -26, and TIMPs-1 and -3 was evaluated by immunohistochemistry in 25 samples of BCC of kidney transplant recipients and 25 matched controls representing superficial and nodular subtypes. No significant differences were detected in MMP expression of BCC tumor cells between immunocompetent and immunodeficient patients. However, MMPs-1 and -9 and TIMP-1 were expressed more frequently in stromal macrophages in the BCCs of immunocompetent patients. When tumor subtypes were compared irrespective of the patient group, more MMP-1-positive fibroblasts and MMP-9-positive neutrophils were detected in the superficial subtype, while stromal MMP-10 expression was more abundant in nodular tumors. Our results suggest that abundant peritumoral expression of TIMP-1 in non-immunocompromised patients limits ECM degradation permissive for cancer cell migration.     

AcSDKP对大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的影响

目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对10%血清和血小板源性生长因子(PDGF)诱导的大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的调节作用。方法明胶酶谱法检测心成纤维细胞MMP-2、MMP-9的活性。Western blot法检测心成纤维细胞MMP-1的表达。结果10%血清和PDGF使心成纤维细胞MMP-2、MMP-9活性增强,也促进MMP-1的表达;AcSDKP能够进一步增加由10%血清和PDGF诱导的心成纤维细胞MMP-2、MMP-9的活性,并促进MMP-1的表达。结论AcSDKP上调了由PDGF介导的心成纤维细胞MMPs活性或表达,这可能与AcSDKP抗心肌纤维化的作用相关。     

Expression of matrix metalloproteinases 2 and 7 in tumor cells correlates with the World Health Organization classification subtype and clinical stage of thymic epithelial tumors

To investigate the roles of matrix metalloproteinases (MMPs) in thymic epithelial tumors, we examined the expression of MMP-2, -7, and -9; membrane-type 1 (MT1)-MMP; and tissue inhibitor of metalloproteinase-2 (TIMP-2) in 57 tumors by immunohistochemistry and in selected 15 cases by in situ hybridization. The tumors consisted of 5 type A, 12 type AB, 11 type B1, 11 type B2, 9 type B3, and 9 type C thymomas according to the World Health Organization histologic classification system and of 22 stage I, 13 stage II, 8 stage III, and 14 stage IV thymomas according to the Masaoka staging system. In the positive cases, MMPs and TIMP-2 were expressed in both tumor cells and stromal cells. The cellular localization of MMPs detected by immunohistochemistry was almost identical with that of the mRNA signals detected by in situ hybridization. MMP-2 and MMP-7 were predominantly expressed in type B3 thymoma and type C thymoma, respectively. Expression of MT1-MMP and TIMP-2 correlated with that of MMP-2, indicating a proteolytic activation of the latter. MMP-9 was prominent in type B2 thymoma. Expression in tumor cells of MMP-2 or MMP-7 was also correlated with clinical stage. The present study suggests that certain MMPs may play an important role in the tumor progression of different subtypes of thymic epithelial tumors and that MMP-2 and MMP-7 may contribute to the tumor aggressiveness and malignant potential.     

MMP-2/MMP-9 plasma level and brain expression in cerebral amyloid angiopathy-associated hemorrhagic stroke

Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Matrix metalloproteinases (MMPs) have been implicated in blood-brain barrier disruption and ICH pathogenesis. In this study, we determined the levels MMP-2 and MMP-9 in plasma and their brain expression in CAA-associated hemorrhagic stroke. Although MMP-2 and MMP-9 plasma levels did not differ among patients and controls, their brain expression was increased in perihematoma areas of CAA-related hemorrhagic strokes compared with contralateral areas and nonhemorrhagic brains. In addition, MMP-2 reactivity was found in β-amyloid (Aβ)-damaged vessels located far from the acute ICH and in chronic microbleeds. MMP-2 expression was associated to endothelial cells, histiocytes and reactive astrocytes, whereas MMP-9 expression was restricted to inflammatory cells. In summary, MMP-2 expression within and around Aβ-compromised vessels might contribute to the vasculature fatal fate, triggering an eventual bleeding.     

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions*

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

MMP-2, MMP-9 and their inhibitors TIMP-2 and TIMP-1 production by human monocytes in vitro in the presence of different forms of hydroxyapatite particles

After calcium-phosphates biomaterials based implantation like hydroxyapatite (HA) coating, particles are released in the periprosthetic tissues. Wear-debris induced fibrous membranes contain macrophage subsets that can produce metalloproteinases (MMPs), which are considered to be key enzymes in extra-cellular matrix turnover. Tissue inhibitors of metalloproteinases (TIMPs) are important regulator of MMPs activity. Interleukin-1 mainly produced by monocytes can also regulate MMPs production. In the present work, we have evaluated the effect of HA particles characteristics (size, shape and sintering temperature) on the MMP-2, -9 and their respective inhibitors TIMP-2, -1 production. Our results demonstrate that sintering temperature (that modify crystal size and surface area) have little effect on MMPs and TIMPs production. Non-phagocytable particles induced more MMP-9, although phagocytable particles induced more IL-1beta release. The shape of the particles was the most important factor since needle-shaped particles induced the most significant up-regulated expression of MMPs and IL-1beta.