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.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Insufficient CD100 shedding contributes to suppression of CD8+ T-cell activity in non-small cell lung cancer

CD100 is an immune semaphorin constitutively expressed on T-cells. Matrix metalloproteinase (MMP) is an important mediator of membrane-bound CD100 (mCD100) cleavage to generate soluble CD100 (sCD100), which has immunoregulatory activity in immune cell responses. The aim of the study was to investigate the level and role of sCD100 and mCD100 in modulating CD8⁺ T-cell function in non-small cell lung cancer (NSCLC). sCD100 and MMP-14 levels in the serum and bronchoalveolar lavage fluid (BALF), and mCD100 expression on peripheral and lung-resident CD8⁺ T-cells were analysed in NSCLC patients. The ability to induce sCD100 and the effect of MMP-14 on mCD100 shedding for the regulation of non-cytolytic and cytolytic functions of CD8⁺ T-cells were also analysed in direct and indirect contact co-culture systems. NSCLC patients had lower serum sCD100 and higher mCD100 levels on CD8⁺ T-cells compared with healthy controls. BALF from the tumour site also had decreased sCD100 and increased mCD100 on CD8⁺ T-cells compared with the non-tumour site. Recombinant CD100 stimulation enhanced non-cytolytic and cytolytic functions of CD8⁺ T-cells from NSCLC patients, whereas blockade of CD100 receptor CD72 attenuated CD8⁺ T-cell activity. NSCLC patients had lower MMP-14 in the serum and in BALF from the tumour site. Recombinant MMP-14 mediated mCD100 shedding from CD8⁺ T-cell membrane, and led to promotion of CD8⁺ T-cell response in NSCLC patients. Overall, decreased MMP-14 resulted in insufficient CD100 shedding, leading to suppression of peripheral and lung-resident CD8⁺ T-cell activity in NSCLC.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in thyroid C-cells and medullary thyroid carcinomas

Aims: Hyperplastic C-cells of the thyroid and medullary thyroid carcinomas (MTCs) were studied immunocytochemically for calcitonin, carcinoembryonic antigen (CEA), chromogranin A, matrix metalloproteinase (MMP)-2 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Methods and results: Non-neoplastic C-cells were positive for all these substances whereas MTC tumour cells were relatively weaker stained, and thyroid follicular cells were negative for all the substances studied. We have recently reported that pancreatic islet cells and islet cell tumours were positive for MMPs and TIMPs, and, in addition, anterior pituitary cells and pituitary adenomas and parathyroid gland and its adenomas were also positively stained. The MMP- and TIMP-positive endocrine cells correspond to Pearse's APUD cells, derived from neural crest and endodermal cells. Conclusions: MMPs and TIMPs may well be added as newly recognized markers for neuroendocrine cells. The possible function of MMP-TIMP homeostasis in C-cells and MTCs is discussed.     

Immunolocalization of MMP-2 and MMP-9 in human rheumatoid synovium

Matrix metalloproteinase (MMP)-2 and MMP-9, two important members of the matrix metalloproteinase family, have been shown critical contributions in intra-tumor angiogenesis and invasion of tumor progression, and they might also play important roles in the angiogenesis as well as the pannus formation of rheumatoid arthritis (RA). In the present study, we used the immunohistochemistry, the immunofluorescence staining and the con-focal scanning methods to characterize the immunolocalization of MMP-2 and MMP-9 in RA synovium tissues. Our results showed that both MMP-2 and MMP-9 immunostaining could be found in synoviocytes and vascular endothelial cells. Moreover, our con-focal scanning also showed that MMP-2 could be found in infiltrating CD14⁺ monocytes and CD68⁺ macrophages, and MMP-9 could be found in infiltrating CD68⁺ macrophages in RA synovium tissues, while weak or negative staining of these two MMPs could be found in infiltrating CD20⁺B cells and CD3⁺T cells in RA synovium. Thus, our finding suggests that both MMP-2 and MMP-9 expressed by synoviocytes as well as certain infiltrating immune cells role importantly in the angiogenesis in RA progression.     

The matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase profile in colorectal polyp cancers

Aims:  The matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system has a major role in tumour invasion and metastasis. Roles in pathways involved in early tumour development are also being identified for this system, and the aim of this study was to define the expression profile of the major MMPs and TIMPs in colorectal polyp cancers.
Methods and results:  The expression and cellular localization of individual MMPs and TIMPs was determined in colorectal polyp cancers by immunohistochemistry. All the MMPs and TIMPs showed immunoreactivity in carcinomatous epithelium. MMP1 ( P  < 0.001), MMP2 ( P  = 0.003), MMP3 ( P  = 0.004), TIMP1 ( P  = 0.01) and TIMP2 ( P  < 0.001) showed significant increases in immunoreactivity in carcinomatous epithelium compared with adenomatous epithelium. MMP7 showed immunoreactivity in carcinomatous epithelium, but showed no immunoreactivity in either normal epithelium or adenomatous epithelium. MMP and TIMP expression was limited in normal epithelium to MMP1, MMP2 and TIMP3.
Conclusions:  This study defines the expression profile of MMPs and TIMPs in colorectal polyp cancers and shows that the increased expression of MMPs and TIMPs occurs at an early stage of colorectal neoplasia. It provides evidence to support the hypothesis that these molecules have a key involvement in the early stages of tumour development.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

CD1a expression defines an interleukin‐12 producing population of human dendritic cells

Human and murine dendritic cell (DC) subsets are often defined by phenotypic features that predict their functional characteristics. In humans and mice, DC have been shown to have the ability to polarize naive CD4 T cells to a T helper type 1 (Th1) or Th2 phenotype. However, human myeloid DC generated from monocytes (monocyte-derived DC) have often been regarded as a homogeneous population, both phenotypically and functionally. Monocytes give rise to subpopulations of DC in vitro that can be separated on the basis of their expression of CD1a, a well-described DC subset marker. Importantly, we show that the CD1a⁺ DC subset produces significant quantities of interleukin-12p70 (IL-12p70) upon stimulation and, similar to the murine CD8α⁺ DC subset, can polarize naive CD4⁺ T cells to a Th1 phenotype. In contrast, CD1a⁻ DC, similar to murine CD8α⁻ DC, do not produce significant amounts of IL-12p70 upon stimulation or polarize T cells to a Th1 phenotype. Like monocyte-derived DC, CD1a⁺ and CD1a⁻ DC subsets obtained from CD34⁺ haematopoietic progenitors under distinct culture conditions were found to have these same features, suggesting that CD1a expression is a marker for myeloid DC that are a major source of IL-12 and Th1 CD4⁺ T cell polarization in man.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

IL‐33 promotes DC development in BM culture by triggering GM‐CSF production

Short‐term DC cultures generated with GM‐CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c⁺ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL‐33 was found to augment DC development time‐ and dose‐dependently. Although the resulting CD11c⁺ cells generated in the presence of IL‐33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL‐12 p70, displayed PD‐L1 and PD‐L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL‐33‐induced expansion of CD11c⁺ cells was completely blocked by anti‐GM‐CSF mAb, and GM‐CSF mRNA and protein expression in BM culture was markedly elevated by added IL‐33, indicating that IL‐33 promotes in vitro DC generation indirectly by a GM‐CSF‐dependent manner. With regard to the cellular source, IL‐33‐dependent GM‐CSF production was observed exclusively within the CD45⁺/FcεRI⁺ BM population. Not only do our results reinforce the notion that GM‐CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in thyroid C-cells and medullary thyroid carcinomas

Aims : Hyperplastic C-cells of the thyroid and medullary thyroid carcinomas (MTCs) were studied immunocytochemically for calcitonin, carcinoembryonic antigen (CEA), chromogranin A, matrix metalloproteinase (MMP)-2 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Methods and results : Non-neoplastic C-cells were positive for all these substances whereas MTC tumour cells were relatively weaker stained, and thyroid follicular cells were negative for all the substances studied. We have recently reported that pancreatic islet cells and islet cell tumours were positive for MMPs and TIMPs, and, in addition, anterior pituitary cells and pituitary adenomas and parathyroid gland and its adenomas were also positively stained. The MMP- and TIMP-positive endocrine cells correspond to Pearse's APUD cells, derived from neural crest and endodermal cells. Conclusions : MMPs and TIMPs may well be added as newly recognized markers for neuroendocrine cells. The possible function of MMP-TIMP homeostasis in C-cells and MTCs is discussed.     

Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

Purpose

Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.

Methods

Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.

Results

At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.

Conclusions

We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.     

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.     

Dysregulation of matrix metalloproteinases and their tissue inhibitors is related to abnormality of left ventricular geometry and function in streptozotocin-induced diabetic minipigs

This study aimed to characterize matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in relation to changes in left ventricle (LV) geometry and function in a porcine model with streptozotocin (STZ)-induced diabetes. In 15 Chinese Guizhou minipigs with STZ-induced diabetes (diabetic group) and 15 age-matched normal controls (control group), Doppler tissue imaging was performed at 6 months of diabetes. Serum MMP-2, -9, TIMP-1, -4 and B-type natriuretic peptide (BNP) were determined. Expression of MMPs, TIMPs, urokinase type-plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in aortic intima and LV myocardium was evaluated, with gelatinolytic activities of tissue MMP-2, -9 accessed by zymography. Left ventricle end-diastolic septum thickness (P < 0.05) and mass (P < 0.05) were increased, whereas peak systolic mitral annulus velocity (Sm, P < 0.001), LV systolic (P = 0.01) and diastolic strain (P < 0.001) were significantly decreased in diabetic group than in controls. Diabetic group showed higher expression of TIMP-1, -4 in aortic intima and LV myocardium (P < 0.01 or P < 0.05), with increased collagen content and elevated serum BNP level (P = 0.004) and lower gelatinolytic activities of tissue MMP-2, -9 (all P < 0.05). Semi-quantitative RT-PCR of those diabetic tissues revealed elevated mRNA levels of major TIMPs, uPA, uPAR and PAI-1. Reduction of serum MMP-2 and -9 levels was observed in diabetic group vs. control group (both P < 0.05). This study features elevated levels of TIMP-1, -4, uPA, uPAR and PAI-1, and decreased activities of MMP-2, -9 in aorta and myocardium in STZ-induced diabetic minipigs, indicating that MMP-TIMP dysregulation is associated with LV hypertrophy, cardiac dysfunction and increased cardiovascular fibrosis in diabetes.     

The role of matrilysin (MMP-7) in leukaemia cell invasion

The matrix metalloproteinases (MMPs) are important in tumour cell invasion and metastasis in many common cancers. However, relatively few studies have investigated the role of MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in leukaemia cell invasion. This study examined two leukaemia cell lines, K562 and HL-60 and showed that the K562 cell line was four times more invasive than the HL-60 cell line. The expression of MMP-2, matrilysin (MMP-7), MMP-9, TIMP-1, TIMP-2 and TIMP-3 was analysed. Both cell lines produced similar amounts of MMP-2, MMP-9 and TIMP-2. The K562 cells expressed more TIMP-1 than the HL-60 cells and neither cell line expressed TIMP-3. Interestingly, only the K562 cells expressed matrilysin suggesting a potential role for matrilysin in leukaemia cell invasion. In vitro invasion assays performed in the presence of a matrilysin blocking antibody showed a 40% reduction in invasive ability. This data suggests that matrilysin plays an important role in leukaemia cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.