In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

MMP and TIMP expression pattern in pleural effusions of different origins

The matrix metalloproteinases (MMP) are proteolytic enzymes that are essentially involved in the turnover of the extracellular matrix (ECM). Their activity is counterbalanced by specific antagonists, the tissue inhibitors of metalloproteinases (TIMP). In this study, we sought to analyze the expression of MMP and TIMP isoforms in pleural effusions from 88 patients. We compared MMP and TIMP isoform expression in transudates (n = 21) and exudates (n = 67), the latter divided into exudates of paraneoplastic (n = 46) or parainfectious (n = 21) origin. Zymographic and Western blot analyses revealed constant expression of interstitial collagenase (MMP-1), gelatinase-A (MMP-2), and TIMP-1 in all 88 samples. In contrast, analyses of gelatinase-B (MMP-9) demonstrated a specific expression pattern, with high expression in exudates and lack of expression in transudates. Neutrophil collagenase (MMP-8) was detected in trace amounts, and correlated with the number of neutrophils in the effusion. Low levels of TIMP-2 were detected only in exudates and not in transudates. Quantitative analysis of the expression ratio of gelatinase-B to gelatinase-A revealed statistically significant differences between effusions of different origin. The ratio was highest in exudates of paraneoplastic origin and lowest in transudates. Our data thus suggest that interstitial collagenase, gelatinase-A, and TIMP-1 play a role in homeostasis of the pleural space in vivo as constitutively expressed proteins, whereas gelatinase-B and TIMP-2 expression are induced in specific disease states. These observations contribute to the understanding of the pathophysiology of pleural effusions, and may help to characterize and possibly distinguish effusions of different origin.     

Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes

PURPOSE: Matrix metalloproteinases (MMP) are a family of extracellular matrix degrading enzymes associated with the development of neovascularization. To investigate the possible role of these enzymes in choroidal neovascularization, the mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) were analyzed in subfoveal fibrovascular membranes from patients with age-related macular degeneration (AMD). METHODS: Surgically removed subfoveal fibrovascular membranes from five eyes were analyzed for the expression of MMP and TIMP mRNA. In situ hybridization anti-sense and sense riboprobes were generated using DNA complementary to human collagenase (MMP-1), 72 kDa gelatinase (MMP-2), stromelysin (MMP-3), 92-kDa gelatinase (MMP-9), TIMP-1, TIMP-2, and TIMP-3. Vascular endothelial cells were detected using immunostaining for von Willebrand factor. RESULTS: MMP-2 and MMP-9 mRNA were detected in all specimens. Most of the membranes also expressed TIMP-1 and TIMP-3 mRNA, and two of the membranes expressed TIMP-2 mRNA. MMP-2, TIMP-1, and TIMP-2 mRNA had a similar overall distribution that was relatively uniform within the vascularized membrane stroma. MMP-2 expression appeared to be localized mainly to the vascular endothelial cells, whereas TIMP-1 and TIMP-3 were detected in other cell types such as fibroblastlike cells. MMP-9 expression was distinctly expressed by cells at the margins of the membranes and often in proximity to a thickened Bruch's membrane-like layer under the retinal pigment epithelial cells. TIMP-3 mRNA was strongly expressed within the retinal pigment epithelial cell layer and also in the stroma of one membrane. None of the membranes showed detectable MMP-1 or MMP-3 expression. CONCLUSIONS: The results support a role for MMPs in the development of choroidal neovascularization in AMD. The localization of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and MMP-9 may be cooperatively involved in the progressive growth of choroidal neovascular membranes in AMD.     

Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis

Asthma and chronic bronchitis are inflammatory diseases with extracellular matrix (ECM) remodeling and collagen deposition. Collagen homeostasis is controlled by metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). We evaluated MMP and TIMP balance in induced sputum of 10 control, 31 untreated asthmatic, and 16 chronic bronchitic subjects. We first performed zymographic analysis to identify the profile of MMPs. Zymography revealed a similar MMPs profile in all populations studied and that MMP-9 was the major enzyme released. We then measured, using enzyme immunoassay, the concentrations of MMP-9 and of its inhibitor TIMP-1 and evaluated whether airflow limitation may be associated with an imbalance between these enzymes. MMP-9 and TIMP-1 concentrations were greater in sputum of patients with asthma and chronic bronchitis than in control subjects. The molar ratio between MMP-9 and TIMP-1 was lower in asthmatics and chronic bronchitics than in control subjects, and positively correlated with FEV1 values. In asthma, MMP-9 levels were significantly correlated with the number of macrophages and neutrophils. This study shows that airway inflammation in asthma and chronic bronchitis is associated with an imbalance between MMP-9 and TIMP-1 which may have a role in the pathogenesis of ECM remodeling and airflow obstruction.     

Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases

In interstitial lung diseases, deposition of extracellular matrix (ECM) in alveoli and degradation of ECM lead to pulmonary structural remodeling. The changes in ECM and the localization of matrix metalloproteinases (MMPs) and a tissue inhibitor of metalloproteinases (TIMP) in the lung tissues of patients with bronchiolitis obliterans organizing pneumonia (BOOP) and idiopathic pulmonary fibrosis (IPF) were investigated. Immunohistochemical analysis for the detection of fibronectin, collagen-I, -III, and -IV, smooth muscle actin, MMP-1 (interstitial collagenase), -2 (gelatinase A), and -9 (gelatinase B), and TIMP-2, and in situ hybridization for the detection of MMP-9 mRNA were performed. Western blotting of lung tissue homogenates was performed for MMP-2 and MMP-9. The gelatinolytic activities of the homogenates were also determined using gelatin zymography. Fibronectin and collagen-I, -III, and -IV were detected in the intra-alveolar fibrosis in addition to the interstitium of these diseases. MMP-1, MMP-2, MMP-9, and TIMP-2 were detected in the regenerated epithelial cells covering intra-alveolar fibrosis. Myofibroblasts in intra-alveolar fibrosis in BOOP showed predominant reaction for MMPs, and they ultrastructurally appeared to be phagocytosing collagen fibrils, and those of IPF showed a predominant reaction for TIMP-2. New vascularization in intra-alveolar fibrosis was exclusively observed in cases of BOOP, and the endothelial cells were positive for MMP-2. Western blotting showed the existence of a latent form of MMP-9 and latent and active forms of MMP-2, and gelatin zymography revealed that the ratio of active/latent forms of MMP-2 in BOOP is significantly larger than that in the control lungs. Predominant MMPs in BOOP may constitute the mechanism of reversibility of fibrotic changes in this disease. TIMP-2 in myofibroblasts in IPF may contribute to the stable ECM deposition and the irreversible pulmonary structural remodeling.     

Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of inflammatory disorders of the central nervous system (CNS) whereas the contribution of the major endogenous counter-regulators of MMPs, the tissue inhibitors of the matrix metalloproteinases (TIMPs), is unclear. We investigated the temporal and spatial expression patterns in the CNS of nine MMP genes and three TIMP genes in normal mice, in mice with EAE, and in transgenic mice with astrocyte (glial fibrillary acidic protein)-targeted expression of the cytokines interleukin-3 (macrophage/microglial demyelinating disease), interleukin-6 (neurodegenerative disease), or tumor necrosis factor-alpha (lymphocytic encephalomyelitis). In normal mice, the MMPs MT1-MMP, stromelysin 3, and gelatinase B were expressed at low levels, whereas high expression of TIMP-2 and TIMP-3 was observed predominantly in neurons and in the choroid plexus, respectively. In EAE and the transgenic mice, significant induction or up-regulation of various MMP genes was observed, the pattern of which was somewhat specific for each of the models, and there was significant induction of TIMP-1. In situ localization experiments revealed a dichotomy between MMP expression that was restricted to leukocytes and possibly microglia within inflammatory lesions and TIMP-1 expression that was observed in activated astrocytes circumscribing the lesions. These findings demonstrate specific spatial and temporal regulation in the expression of individual MMP and TIMP genes in the CNS in normal and inflammatory states. The distinct localization of TIMP-1 and MMP expression during CNS inflammation suggests a dynamic state in which the interplay between these gene products may determine both the size and resolution of the destructive inflammatory focus.     

Low protein diet blunts the rise in glomerular gene expression in focal glomerulosclerosis

The present study was designed to assess whether expression of mRNA for extracellular matrix (ECM) components, metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) in glomeruli is affected by a low protein diet during the course of focal glomerulosclerosis (FGS). Puromycin aminonucleoside (PAN) was injected intraperitoneally in rats and the right kidney was removed on day 22. Nephrotic rats received successive intraperitoneal injections of PAN on days 27, 34, and 41. Control rats were subjected to a nephrectomy or a sham operation on day 22. Animals were divided into six groups. In group 1, the PAN-injected rats were fed a standard diet containing 22% protein. In group 2, the PAN-injected rats were fed a low protein diet containing 6% protein, starting on the same day as the first PAN injection. In group 3, the nephrectomized rats without PAN were fed a standard diet. In group 4, the nephrectomized rats without PAN were fed a low protein diet for the same period. In group 5, the sham operated rats were fed a standard diet. In group 6, the sham operated rats were fed a low protein diet for the same period. Rats were sacrificed on days 0, 60 or 80 after the initial PAN or saline injection. The percentage of sclerotic glomeruli in group 1 rats increased markedly with time, reaching 77% on day 80. The mRNA levels encoding for alpha 1(I), alpha 1(III), alpha 1(IV) collagen chains, laminin B1 and B2 chains, heparan sulfate proteoglycan (HSPG), MMP-2, TIMP-1 and TIMP-2 increased significantly as glomerulosclerosis progressed, whereas MMP-1 and MMP-3 mRNA levels were unchanged, and no MMP-9 mRNA was detected throughout the experiments. In group 2, the low protein diet reduced the prevalence of glomerulosclerosis and attenuated the increased mRNA expression for ECM components, MMP-2, TIMP-1 and TIMP-2 in FGS glomeruli. In groups 3 through 6, mRNA levels for ECM components decreased with age, whereas those for MMPs and TIMPs changed little throughout the experiments. Immunofluorescence studies revealed the accumulation of types I, III and IV collagens, laminin, and HSPG in the sclerotic area and low protein diet attenuated the accumulation of these proteins. These data suggest that glomerulosclerosis may result from an imbalance among ECM components, MMPs and TIMPs and that a low protein diet attenuates the otherwise increased levels of mRNA for ECM components, MMP-2, TIMP-1 and TIMP-2 in glomerulosclerosis.     

Regulation of matrix-degrading enzymes in gynecologic cancer tissues and cells

1. INTRODUCTION: Studies of tumor invasion and metastases have focused on the degradation of the basement membrane, which is predominantly made up of type IV collagen, laminin, and heparan sulfate proteoglycans. Matrix metalloproteinase-2 (MMP-2) and MMP-9, which can degrade type IV collagen, are implicated in cancer invasion and metastasis. Released and activated MMPs are controlled by specific tissue inhibitors of metalloproteinase (TIMP). In the present study, we have examined gelatinolytic and TIMP activity in the conditioned medium of human normal and cancer tissues by zymography and reverse zymography. 2. MATERIALS AND METHODS: 1) Tissues. Tissues were obtained at operation after informed consent was got from each patient. Sliced tissues were incubated in serum-free medium for 4 or 24 h at 37 degrees C. Human ovarian cancer cells (SAOV) were cultured for 24 h in serum-free medium containing conditioned medium of stromal tissues. After washing by PBS 3 times, SAOV cells were cultured for a further 24 h. 2) Zymography. Conditioned medium was subjected to SDS polyacrylamide gel containing 0.3 mg/ml of gelatin in zymography, and purified MMPs were added further in reverse zymography. After electrophoresis the gel was washed with Triton X-100, and incubated for 20 h at 37 degrees C in the reaction buffer. The gel was then stained with Coomassie brilliant blue. The gelatinase and TIMP activities were detected as unstained and stained bands, respectively. The photographs of the gels were scanned with a densitometer. 3) Other method. TIMP-1 levels of conditioned medium were assayed by ELISA kit. 4) Statistics. Statistical comparisons were made by Mann-Whiteny U test. 3. RESULTS AND DISCUSSION: We have examined the gelatinolytic activity in gynecologic normal and cancer tissues by zymography and reverse zymography. Ovarian, cervical, and endometrial cancer tissues demonstrated higher gelatinolytic activity than normal tissues. The major gelatinases were those with molecular weight of 92 and 72kD, which corresponded to MMP-9 and MMP-2, respectively. The ratio of MMP 9 to MMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. Reverse zymography demonstrated that TIMP-1 and TIMP-2 were present in all tissues, and the ratio of TIMP-1 to TIMP-2 was significantly higher in 3 types of cancer tissues than in normal tissues. These findings suggested that MMP-9 and TIMP-1 were more associated with cancer phenotype than other types of MMP and TIMP. The influence of human stromal tissues (peritoneum, myometrium, ovary) on the secretion of MMPs and TIMPs was examined by addition of these stromal tissues culture medium to human ovarian cancer cells (SAOV). All conditioned medium of stromal tissues could increase in both MMP-2, MMP-9, TIMP-1, and TIMP-2 activity in SAOV cells. Fraction (> 100kD) of conditioned medium of peritoneum could increase remarkably in MMP-9, and this increase could be inhibited by anti alpha 5 antibody, which is the most popular receptor of fibronectin. Furthermore, the addition of fibronectin to SAOV cells induced increase in the secretion of MMP-9. These results demonstrated that one of the factors included in conditioned medium of peritoneum was fibronectin. We found that interferon beta could suppressed the secretion of MMP-2 and invasion in choriocarcinoma cells. However, no effect of interferon beta was observed in SAOV cells. Several flavonoids were screened to have ability to suppress the secretion of MMPs. All trans retinoic acid (RA) could suppress the secretion of MMPs in SAOV cells in time and concentration dependent manners. Further, RA could inhibited the invasion of SAOV cells by invasion assay using boyden chamber coated with matrigel.     

The effect of luteal "rescue" on the expression and localization of matrix metalloproteinases and their tissue inhibitors in the human corpus luteum

Luteolysis is associated with tissue remodeling probably involving the matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs). This study investigated the expression and localization of the major MMPs and TIMPs in the human corpus luteum throughout the luteal phase and after luteal rescue with hCG. Corpora lutea (n = 9) were collected at hysterectomy and were dated by serial urinary LH estimation. In addition, corpora lutea (n = 3) were collected from women who had received daily doubling doses of hCG to mimic the hormonal changes of early pregnancy. MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were investigated by zymography, reverse zymography, Northern blotting, and in situ hybridization. There was no change in the expression of MMP-1, TIMP-1, and TIMP-2 throughout the luteal phase or after luteal rescue. Little TIMP-3 could be detected in the corpus luteum. MMP-9 activity peaked in the early and late luteal phase. The expression and activity of MMP-2 were maximal in the late luteal phase. Exposure to hCG during luteal rescue in vivo was associated with a reduction (P < 0.05) in the expression and activity of MMP-2. Messenger ribonucleic acids (mRNAs) for MMP-1, MMP-2, and TIMP-2 were localized to the connective tissue stroma and the thecal-lutein cells of the corpus luteum. In contrast, TIMP-1 mRNA was localized to the granulosa-lutein cells, and MMP-9 mRNA was expressed in scattered cells within the steroidogenic and nonsteroidogenic cell layers. In conclusion, during maternal recognition of pregnancy, hCG prevents the normal increase in MMP-2 in the late luteal phase. MMPs can function in an environment containing large amounts of TIMP-1, as they have a different cellular localization.     

Gene expression of MMPs and TIMPs in the process of hepatic fibrosis

The matrix metalloproteinases (MMPs) gene family includes MMP-1 (interstitial collagenase), MMP-2 (72 kD type IV collagenase/gelatinase), MMP-3 (stromelysin/transin), MMP-7 (putative MMP; pump-1), MMP-8 (granulocyte collagenase) and MMP-9 (92 kD type IV collagenase/gelatinase). This gene family has the common characteristics in the gene structure as follows: All of MMPs have the active site metal ion-binding domain. All six enzymes are activated with the concomitant removal of N-terminal segment of the latent enzyme. The removed segment contains an unpaired cystein residue within the conserved amino acid sequence PRCGVPDV, located immediately adjacent to the proenzyme cleavage site. The authors showed the gene expression of MMP-1 in the process of hepatic fibrosis. The remarkable expression was noted on fibroblasts and macrophages within the newly-formed fibrous bands with lots of infiltrated lymphocytes. Liver cirrhosis did not showed the positive dots of MMP-1 mRNA. On the other hands, the expression of TIMP reported by Takahara et al., revealed the high level of expression in the advanced fibrosis.     

Bi-directional induction of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 during T lymphoma/endothelial cell contact: implication of ICAM-1

The mechanisms that lead to the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMPs) during the invasive process of normal and transformed T cells remain largely unknown. Since vascular cells form a dynamic tissue capable of responding to local stimuli and activating cells through the expression of cytokine receptors and specific cell adhesion molecules, we hypothesized that the firm adhesion of T lymphoma cells to endothelial cells is a critical event in the local production of MMP and TIMP. In the present work, we show that adhesion of lymphoma cells to endothelial cells induced a transient and reciprocal de novo expression of MMP-9 mRNA and enzymatic activity by both cell types. Up-regulation of MMP-9 in T lymphoma cells was concomitant to that of TIMP-1, and required direct contact with endothelial cells. Induction of MMP-9, but not of TIMP-1, was blocked by anti-LFA-1 and anti-intercellular adhesion molecule-1 Abs, indicating that induction of MMP-9 and TIMP-1 in lymphoma cells required direct, yet distinct, intercellular contact. In contrast, the induction of MMP-9 in endothelial cells by T lymphoma cells did not necessitate direct contact and could be achieved by exposure to IL-1 and TNF, or to the supernatant of T lymphoma cell culture. Together, these results demonstrate that firm adhesion of T lymphoma cells to endothelial cells participates in the production of MMP-9 in both cell types through bi-directional signaling pathways, and identify intercellular adhesion molecule-1/LFA-1 as a key interaction in the up-regulation of MMP-9 in T lymphoma cells.     

Clinical and experimental studies of JPYS in reducing side-effects of chemotherapy in late-stage gastric cancer

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective-tissue-matrix remodelling and the accelerated breakdown associated with tumor development. These MMPs and tissue inhibitor of MMPs (TIMP1) could be expressed by either the cancer or the stromal cells. Expression of mRNAs encoding interstitial collagenase (MMP1), 72-kD type IV collagenase (MMP2) and stromelysin (MMP3), which are probably involved in tumor invasion and metastasis, and of TIMP1 were studied in human mammary pathology by in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells. These data suggest that there is a cooperation between tumor and stromal cells, in particular for the production of 72-kD type IV collagenase, involved in the disruption of basement membranes. A lack of TIMP1 expression from invasive cancer cells would also contribute to matrix destruction.