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.     

Factors inducing mast cell accumulation in skeletal muscle

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean +/- SEM/mm2) range from 1.8 +/- 1 to 4.3 +/- 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5 +/- 13.0 vs 12.3 +/- 1.8/mm2). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is though to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.     

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.     

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Human anti-Ro autoantibodies bind multiple conformational epitopes of 60-kD Ro autoantigen

Bronchial asthma is characterized by eosinophil infiltration and tissue remodeling. Matrix metalloproteinases (MMPs) are thought to play critical roles by degradating interstitial matrices in a wide range of lung diseases associated with reorganization of the airway architecture. To investigate whether MMPs are involved in the pathologic processes of bronchial asthma, we examined MMP expression in asthmatic subjects. In situ hybridization revealed abundant expression of MMP-9 (gelatinase B) mRNA in biopsy specimens from asthmatic subjects (n = 5), with an average positive cell distribution of 117.8 +/- 41.1 (mean +/- SEM)/mm2. In contrast, sparse expression of the mRNA (10.8 +/- 4.8 /mm2) was observed in specimens from normal subjects (n = 4). The vast majority of cells expressing the mRNA were eosinophils in asthmatic tissues (92.2 +/- 1.2%). MMP-9 protein, which was confined to the submucosal cells in the normal subjects, was not abundantly expressed in inflammatory cells, but there was positive reactivity for MMP-9 protein in the extracellular matrix. Immunoelectron microscopic analysis showed sparse immunolocalization of MMP-9 in the perinuclear spaces of eosinophils, but not in the granules. These findings suggest the overexpression of MMP-9 by eosinophils in bronchial tissues of asthmatic individuals, and the participation of MMPs in the pathologic changes in asthmatic airways.     

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)     

Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.     

Insulin resistance and clinical evolution in infantile myotonic dystrophy

1. Extensor digitorum longus muscles of C57 BL/10 and mdx mice were overloaded by removing the synergist tibialis anterior muscle of 9-12-day-old animals. The effect of this operation on the weight, contractile properties and force of the extensor digitorum longus muscle was examined at two different ages, i.e. at 2-3 months (young group) and at 5-8 months (old group). The changes with age in both the control and overloaded muscles of normal and mdx mice are also described. The values obtained from the overloaded muscles were always compared with those for the control, unoperated extensor digitorum longus. 2. In the normal strain of mice the weight of the overloaded extensor digitorum longus muscle in the younger group was increased and it remained higher in the older animals. In the mdx mice the overloaded extensor digitorum longus muscles weighed more in the younger animals but not in the older group of mice. 3. The twitch and tetanic tensions of the overloaded muscles were slightly, but not significantly, increased in the younger group of mdx mice, whereas in the older animals there was a significant decrease in both twitch and tetanic tensions. 4. Thus the overloaded muscles from mdx mice progressively deteriorated with age. In both strains of mice the overloaded muscles become less fatigable with time.     

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.     

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.     

Magnetic resonance imaging of regenerating and dystrophic mouse muscle

Magnetic resonance imaging allows serial visualization of living muscle. Clinically magnetic resonance imaging would be the first step in selecting a region of interest for assessment of muscle disease state and treatment effects by magnetic resonance spectroscopy. In this study, magnetic resonance imaging was used to follow dystrophy and regeneration in the mdx mouse, a genetic homologue to human Duchenne muscular dystrophy. It was hypothesized that images would distinguish normal control from mdx muscle and that regenerating areas (spontaneous and after an imposed injury) would be evident and evolve over time. T2-weighted images of hind-limb muscles were obtained on anaesthetized mice in a horizontal bore 7.1-T experimental magnet. Magnetic resonance images of mdx muscle appeared heterogeneous in comparison to homogeneous images of control muscle. Foci of high intensity in mdx images corresponded to dystrophic lesions observed in the histologic sections of the same muscles. In addition, it was possible to follow chronologically the extent of injury and repair after an imposed crush injury to mdx muscle. These results should make it possible to obtain meaningful magnetic resonance spectra from particular regions of interest in muscle as viewed in magnetic resonance images (i.e., regenerating, degenerating, normal muscle) acquired during neuromuscular diseases and treatment regimens.     

Apoptotic myonuclei in human Duchenne muscular dystrophy

The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibers of the mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in dystrophin-deficient mice 2 days after spontaneous exercise. To investigate the role of apoptosis in human muscular dystrophy, muscles from 11 patients of different ages with Duchenne muscular dystrophy were analyzed for apoptosis. The amount of apoptosis was assessed by terminal deoxynucleotidyl transferase assay, and the expression of bcl-2 and bax was examined by immunohistochemistry. Although very rare in normal muscles (less than 0.1%), apoptotic nuclei were detected in dystrophic muscles, particularly at the interstitial level. Nevertheless, few dystrophin-deficient myofibers with centrally located nuclei showed a positive reaction for DNA fragmentation. A mosaic pattern of bcl-2/bax-positive myofibers characterized dystrophic muscles, thus the relative proportion of pro- and antiapoptotic proteins differs among muscle fibers in correlation with the presence of apoptotic myonuclei. In the interstitium, apoptotic cells were identified as macrophages and activated satellite cells. This is the first study to show an apoptotic process in adult muscle fibers of patients with Duchenne muscular dystrophy, thereby shedding new light on muscle damage and its progression in dystrophinopathies.     

Diagnosis of lumbar disc herniation by three-dimensional MRI

Trophoblasts cells which are derived from the outer layer of the blastocyst have developed mechanisms by which they can invade the uterus and tap into the maternal circulation. In contrast to tumor cell invasion trophoblast invasion is precisely regulated, being confined spatially to the uterus and temporally to early pregnancy. The invasive properties manifested by trophoblasts are made possible by the secretion of proteolytic enzymes which can degrade components of the extracellular matrix (ECM). A number of investigators have shown that the matrix metalloproteinases (MMPs) are important mediators of trophoblast invasion. The two type IV collagenases, MMP-2 and MMP-9, which specifically degrade type IV collagen and gelatins have been of particular interest in this respect. In this paper we examine the expression and regulation of MMPs and their inhibitors in a series of trophoblast continuous cell lines. These cell lines, ED27, ED31, ED77, and a choriocarcinoma cell line, BeWo, were initially characterized with respect to various properties, including cytokeratin, hCG, and hPL expression. We have looked at the expression of MMPs and their inhibitors in these cell lines and their in vitro invasive behavior. Using zymography and RT-PCR we show that the trophoblast cell lines produce both MMP-2 and MMP-9, while the BeWo produce only MMP-2. Using an in vitro invasion assay the trophoblast cell lines were shown to be capable of invading while the BeWo were unable to invade. These results suggest that expression of MMP-9 in these cells is crucial for invasion. We have also examined the regulation of MMP expression by cytokines and found that MMP-9 expression could be modulated by IL-1 beta in these cell lines. The data presented in this paper suggest that these trophoblast cell lines present an ideal model system to investigate the regulation of metalloproteinases in trophoblast invasion.     

Dystrophin-positive muscle fibers following C2 myoblast transplantation into mdx nude mice

To determine when and how the dystrophin-positive muscle fibers are formed after myoblast transplantation into dystrophin-negative muscles, the tibialis anterior (TA) muscle from mdx nude mouse was chronologically examined after C2 myoblast transplantation by immunohistochemical and glucose 6-phosphate isomerase (GPI) isoenzyme analyses. The host TA muscle transplanted with C2 myoblasts became necrotic with accumulation of basic fibroblast growth factor in the necrotic areas. This may stimulate concomitant proliferation of the host satellite cells and C2 myoblasts. Small dystrophin-positive muscle fibers appeared in the necrotic areas 3 days after transplantation. This TA muscle contained two different kinds of homodimer GPI isoenzymes but did not contain the heterodimer, suggesting rare fusion of host and donor cells. The dystrophin-positive muscle fibers in the necrotic areas rapidly increased in number and in size by 7 days, but they were smaller than the original host muscle fibers. They had central nuclei, indicating that they were regenerating fibers. The presence of heterodimer GPI isoenzyme in these muscles indicated that the regenerating fibers were mosaic host/donor muscle fibers. The dystrophin-positive muscle fibers are probably formed first by fusion of donor cells with each other and then later by the fusion of host satellite and donor cells.     

Evaluation of a western blot system (DAVIH-BLOT) for the confirmation of HIV-1 antibodies]

Matrix metalloproteinases (MMPs), a sort of important enzymes involved in extracellular matrix metabolism, play critical roles in the process of tissues remodeling, wound healing and metastasis of tumors. Dot blot and in situ hybridization were used in this study to detect the expression and localization of MMP-9, an important proteolytic enzyme implicated in bone resorption in bone tissues. The results showed that the level of MMP-9 mRNA expression in osteoporotic bone tissues was significantly higher than that in normal control group and the cell types that expressed MMP-9 mRNA included mono- and multi-nuclear osteoclasts and some lining cells on the surface of bone matrix. It was suggested that MMP-9 play a key role in the development of bone loss in osteoporosis.     

Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific

OBJECTIVES: Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA. METHODS: Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with lambda DNA. Rheumatoid synovium was used as a positive control for MMP gene expression. RESULTS: Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease. CONCLUSION: The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.     

Melatonin chimeras alter reproductive development and photorefractoriness in Siberian hamsters

Apoptosis is well accepted as a type of cell death occurring in the development of mammalian muscles, but the death of adult myofibres in neuromuscular disorders and exercise-induced muscle damage is usually explained in terms of muscle necrosis. The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibres of mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in mdx mice 2 days after spontaneous exercise. To investigate the contribution of apoptosis to exercise-induced damage of normal muscle fibre a time-course analysis has been performed in adult C57BL/6 mice. Groups of five mice were sacrificed immediately after the end of the exercise, and after a rest period of 6 or 96 h. The amount of apoptosis in leg muscles was assessed by electron microscopy, by the terminal deoxynucleotidyl transferase assay and by electrophoretic detection of fragmented DNA; the expression of Bcl-2, Bax, Fas, ICE, p53 and ubiquitin was examined by immunohistochemistry and Western blot. Absent in muscles of normal 'sedentary' mice, apoptotic myonuclei peak in muscles of normal mice after a night of spontaneous wheel-running (4% +/- 3.5, immediately and 2.5% +/- 1.8 after 6 h rest, P < 0.05 vs non-runner mice); they then decrease but are present 4 days later (0.8% +/- 1.5). Satellite cells are also involved in the apoptotic process. Myofibre content of Bcl-2 decreases whereas Bax, Fas, ICE and ubiquitin modify their pattern of expression in correlation with the changes in apoptotic myonuclei. Apoptosis of endothelial cells is present after the night of wheel-running and with a twofold increase 4 days later (1.5 +/- 2.3 and 4.8 +/- 4.4 P < 0.05, respectively). Satellite cells are also involved in the apoptotic process. Thus, spontaneous running in unaccustomed mice increases the number of apoptotic nuclei in adult muscle fibres and in endothelial cells. It remains to be established whether muscle apoptosis is restricted to the repair mechanisms, as often suggested in many pathologic processes, or it is also part of pathogenesis of muscle damage. Regardless of whether these results are extended to human dystrophies, the clinical implications in terms of secondary pathogenetic mechanisms and muscle training are obvious.     

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.