[The assay of tissue collagenase activity using fluorescein isothiocyanate labeled collagen].

This report describes the assay of collagenolytic activity, particularly activity of tissue collagenases, using the reconstituted fibrils of fluorescein-labeled collagen type I as substrate. Labeling of soluble rat skin collagen was carried out by the modified method of Baichi et al 1980. This method is very sensitive, reproducible and useful for screening large number of samples.

A pilot study identifying type V collagenolytic activity in human amniotic fluid.

Amniorrhexis complicates pregnancies if it occurs in a preterm pregnancy or remote from the onset of labor in a term pregnancy. There are different collagen types (I-V) that create the extracellular matrix of the amnion. Collagenases specific to these collagen types, with the exception of type V collagen, are found in human amniotic fluid, fibroblasts, polymorphonuclear leukocytes and bacteria. Type V collagen is a major component of the amniotic basement membrane and is responsible for maintaining a barrier to bacteria and to the loss of amniotic fluid. We sought to find evidence of type V collagenolytic activity in human amniotic fluid obtained from pregnancies in different clinical states.

Borrelia burgdorferi possesses a collagenolytic activity.

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi, an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.

The role of mucin in colon-cancer metastasis.

Mucinous colorectal cancer often presents at an advanced stage. We have previously observed that mucin production by human colon-cancer cells correlates with their ability to colonize the liver in experimental animal models. The present study was undertaken in order to further elucidate the mechanisms by which production of mucin by colon-cancer cells affects metastasis. Cell lines showing high mucin production (HMP) (HM 7, HM 3 and LS LiM 6) demonstrated increased adherence to basement membrane proteins and invaded a reconstituted basement membrane to a greater extent than their counter-part cell lines showing low mucin production (LMP) (LS174T and LM 12). Adherence of the LMP parental cell line LS174T to various matrix proteins was potentiated by the addition of purified human colon-cancer mucin in a dose-dependent fashion. HMP cell lines secreted more proteolytically active type-IV collagenase than LMP lines, and collagenase activity was further stimulated by purified mucin in a dose-dependent manner. Specific inhibition of mucin O-glycosylation by benzyl-alpha-N-acetylgalactosamine significantly affected each of the metastasis-related events, with the greatest effect on the HMP cell lines. The present data further indicate that mucin may play an important role in the metastatic process.

Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen alpha 1 (IV) chain genes by cells of early human placenta and gestational endometrium.

BACKGROUND: In this study we used in situ hybridization to investigate the expression of the genes 70 kilodalton (kd) collagenase and the alpha 1(IV) collagen chain of type IV collagen in cells of early human placenta and gestational endometrium. EXPERIMENTAL DESIGN: The aim was to study the spatial distribution of these gene expressions within a developing tissue which possesses physiologic invasive potential. The results obtained for the 70 kd type IV collagenase mRNA expression were also compared with the immunohistochemical distribution of the corresponding antigen. RESULTS: Expression of mRNAs for these proteins was found in cells of trophoblastic columns, stromal cells of villi and in cells of decidua and endometrial stroma. The only differences between the expressions was the lower level of signals for 70 kd type IV collagenase in fibroblastic stromal cells and endothelial cells of villi and in the pericytic cells of spiral arteries. Otherwise the results for both types of mRNA were comparable. We also studied the immunohistochemical distribution of the 70 kd type IV collagenase using specific monoclonal antibodies against the enzyme. Immunohistochemistry supported well the findings obtained by in situ hybridization. CONCLUSIONS: The results indicate that the genes for the 70 kd type IV collagenase and for the alpha 1(IV) collagen chain are simultaneously active in cells of placenta and gestational endometrium and the same cells which produce type IV collagen also can produce the cleaving enzyme, the 70 kd type IV collagenase. The results also show that the cytotrophoblastic cells, which during early pregnancy invade the extracellular matrix and spiral arteries of uterine wall contain significant amount of mRNA for the 70 kd type IV collagenase. This finding supports the concept that the 70 kd type IV collagenase would be important for invasion, and in the case of this study, also for the physiologic invasion of placental cytotrophoblasts.

Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis.

Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.

Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction.

Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease.

Laminin in the human embryo implantation: analogy to the invasion by malignant cells.

OBJECTIVE: To explore the possible similarities between the biochemical processes of embryo implantation and malignant invasion. DESIGN: The expression of a basement membrane (BM) glycoprotein laminin, a matrix binding cell surface receptor protein beta 1-integrin, and a BM collagen degrading metalloproteinase type IV collagenase, was studied in cultured human in vitro fertilized embryos. PATIENTS: Eight healthy women suffering from tubal infertility were participating in the IVF program in the Department of Obstetrics and Gynecology in University Hospital of Oulu. Twenty oocytes and 110 pre-embryos that were not transferred for the fertilizations were used in this study. MAIN OUTCOME MEASURES: Fibronectin, laminin, beta 1-integrin, and type IV collagenase immunoreactive proteins were studied in embryos by immunoperoxidase staining, and type IV collagen degrading activity was measured from the culture media of the embryos. RESULTS: Laminin and beta 1-integrin were expressed in the early human embryos before the time of implantation. Type IV collagen degrading activity and the 72 kd-type IV collagenase immunoreactive protein were expressed at the time of implantation. Laminin supported the expression of type IV collagenase. CONCLUSIONS: The expression of laminin, beta 1-integrin, and type IV collagenase in vitro are temporally in good correlation with the time of the implantation in vivo. Laminin and beta 1-integrin can relate to the attachment of the embryos to the uterine BM and type IV collagenase to the degradation of the BM collagen during the implantation. Laminin can augment the process locally.

Immunohistochemical profile of basement membrane proteins and 72 kilodalton type IV collagenase in the implantation placental site. An integrated view.

An immunohistochemical study was performed to investigate the interactions between trophoblast and the extracellular matrix in the implantation site of early pregnancies. Two basement membrane-related proteins (type IV collagen and laminin), as well as the expression of the 72 kilodalton type IV collagenase, were studied with affinity-purified antibodies. human placental lactogen, human chorionic gonadotropins, and AE1/AE3 cytokeratins were used to identify the different cell populations involved in the implantation process. All types of trophoblastic cells, from villous cells to the different types of intermediate trophoblast, expressed the 72 kilodalton type IV collagenase. Decidual cells, Hofbauer's cells, villous fibroblasts, and amnion were also positive. Laminin and type IV collagen were expressed in all basement membranes, including large decidual and intermediate trophoblast cells, and the villous stroma. Nitabuch's layer, an acellular degradative zone at the site of initial attachment, showed positivity for type IV collagen. The extracellular matrix in the implantation site seems to be a meshwork of, among other components, laminin and type IV collagen, in which the invading trophoblastic cells are embedded. The invasive capacity of these cells in vivo may be, at least in part, mediated by their type IV collagenolytic activity along with that of the decidual cells, thus regulating the permeability of the extracellular matrix.

Expression of gelatinase/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion.

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhance in vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinase/type IV collagenase in NE. Moreover, NE increased the in vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinase/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.

Evaluation of basement membrane components and the 72 kDa type IV collagenase in serous tumors of the ovary.

We studied the distribution of the basement membrane components laminin and type IV collagen in 46 serous tumors of the ovary, including a group of low malignant potential tumors with microinvasion. The findings were correlated with the expression of the 72 kDa type IV collagenase, an enzyme that initiates the degradation of type IV collagen and consequently may play a role in the process of invasion. Benign cystadenomas and tumors of low malignant potential without microinvasion showed a continuous basement membrane; whereas invasive carcinomas, peritoneal implants, and lymph node metastasis had frequent disruptions and extensive areas without basement membrane components. Early invasion in tumors of low malignant potential was characterized by focal disruptions in basement membranes and complete absence of laminin and type IV collagen around single or clusters of microinvasive cells. Type IV collagenase was negative or minimally expressed in cystadenomas, whereas in invasive carcinomas and metastasis the reactivity was moderate to intense. Microinvasive cells in tumors of low malignant potential were strongly positive. The collagenase IV was also localized in cell clusters elsewhere in the tumors where the basement membrane was still preserved. These cells had a similar morphology to that of the microinvasive cells. We conclude that detection of basement membrane components may be useful in recognizing early invasion in this group of ovarian neoplasms. The correlation between progressive anomalies of the basement membrane and expression of type IV collagenase suggests that this enzyme functions directly in the degradation of basement membrane components and facilitates the invasive process.

Collagen loss in the stunned myocardium.

BACKGROUND: This study was performed to biochemically assess and quantify the previously observed ultrastructural alterations in the collagen matrix of stunned myocardium. METHODS AND RESULTS: The stunned myocardium was produced in 13 mongrel dogs by a series of 12 coronary artery occlusions of 5 minutes followed by 10-minute reperfusion periods, with a final reperfusion period of 90 minutes. Regional systolic function in the stunned myocardium was 17% of control. Relative end-diastolic length in the stunned region increased up to 8%. There was a nonuniform transmural loss of collagen. Hydroxyproline in the stunned endocardium was not different from control. The stunned midwall and epicardium demonstrated 12.5% (p less than 0.05) and 14.6% (p less than 0.005) decreases, respectively. All transmural layers in the stunned myocardium had significant increases in collagenase activity before procollagenase activation, averaging a 73.6% increase (p less than 0.025). Complete activation of all procollagenase forms with aminophenylmercuric acetate revealed no differences in fully activated collagenase between the stunned and normal regions. The lysosomal enzymes, elastase and cathepsin G, were not different between stunned and normal zone tissue. These results would tend to exclude exogenous sources of protease in the stunned myocardium at the 90-minute final reperfusion time frame. Collagen fibers were isolated from the stunned and normal zone tissue and underwent dansyl chloride reaction. Stunned collagen fibers had 9% greater dansyl labeling, suggesting greater numbers of exposed N-terminal amino acid residues on the fiber and compatible with greater enzymatic cleavage activity on the stunned collagen matrix. Tissue water content was consistently greater in the stunned region compared to the normal: a uniform transmural increase of approximately 1.7%. CONCLUSIONS: The stunned myocardium is characterized by both systolic dysfunction and diastolic expansion or dilatation. Endogenous procollagenase is activated by the ischemic process leading to degradation of the extracellular matrix. The underlying mechanisms may be relevant in ischemic enlargement of the heart and cardiomyopathy.

Tetracycline inhibition identifies the cellular origin of interstitial collagenases in human periodontal diseases in vivo.

Mammalian interstitial collagenases (E.C.3.4.24.7) are considered as key initiators of collagen degradation in periodontal diseases. However, the cellular sources of collagenases present in gingival crevicular fluid have not been completely clarified. Resident fibroblasts and epithelial cells as well as infiltrating neutrophils and monocyte/macrophages are potential sources of the enzymes. We have recently found significant differences in tetracycline inhibition between human neutrophil and fibroblast interstitial collagenases. To address the cellular source of collagenase present in gingival crevicular fluid in 2 distinct periodontal diseases, we studied the tetracycline inhibition of collagenase in gingival crevicular fluid of patients with localized juvenile periodontitis and adult periodontitis. Gingival crevicular fluid samples were collected from deep (greater than 5 mm) periodontal pockets and assayed for collagenase in the presence of 0-1000 microM doxycycline as well as a chemically modified tetracycline devoid of antimicrobial activity (4-de-dimethylaminotetracycline). The drug concentration required to inhibit 50% of collagenase activity (IC50) in localized juvenile periodontitis gingival crevicular fluid was 280 microM for doxycycline and 470 microM for 4-de-dimethylaminotetracycline. Significantly lower values, 10-20 microM, were obtained for collagenase in gingival crevicular fluid of patients with adult periodontitis. We propose that systemic tetracycline levels are efficient inhibitors of collagenase in gingival crevicular fluid in affected sites of patients with adult periodontitis but not of patients with localized juvenile periodontitis and that the fibroblast type interstitial collagenase is the predominant collagenase type in gingival crevicular fluid in affected sites of patients with localized juvenile periodontitis and the neutrophil collagenase in adult periodontitis gingival crevicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of collagenase and potential transcriptional factors in the MRL/l mouse arthropathy.

Typical erosions of articular joint structures in rheumatoid arthritis and in the spontaneous destructive hind-limb arthropathy of autoimmune MRL-lpr/lpr (MRL/l) mice occur predominantly in areas contiguous with proliferating synovial lining cells, suggesting release of proteolytic enzymes from these cells. Synovial lining cells were isolated from arthritic MRL/l mice, and the spontaneous expression of the interstitial procollagenase and its potential transcriptional factors, egr-1 and c-fos, was examined in vitro. The data indicate that basal collagenase RNA expression was stronger in MRL/l cells than in virus-transformed cells. Moreover, elevated RNA levels of the c-fos gene could be detected in the collagenase-expressing synovial lining cells in vitro. In a related immunohistochemical study, collagenase was detected in situ in proliferating synovial lining cells as well as in chondrocytes of the first stage of pathological changes in the MRL/l mouse arthropathy.

Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential.

BACKGROUND: Several protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas. PURPOSE: Our purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior. METHODS: We evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis. RESULTS: In A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblotting, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070-10 and C1070-14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity. CONCLUSION: These findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines.

Detection of stromelysin and collagenase in synovial fluid from patients with rheumatoid arthritis and posttraumatic knee injury.

OBJECTIVE: To quantify stromelysin and collagenase in synovial fluid (SF) from patients with rheumatoid arthritis (RA) or traumatic knee injury. METHODS: Stromelysin and collagenase were measured in the SF of 33 patients with RA or posttraumatic knee injury, using specific double-antibody sandwich enzyme-linked immunosorbent assays. Stromelysin was fractionated from representative SF, and the molecular form was identified by immunoblot analysis. RESULTS: The stromelysin concentration was approximately 20-fold higher than the collagenase concentration in the fluids from patients with RA and approximately 8-fold higher in the fluids from patients with traumatic injury. For both metalloproteinases, there was a higher enzyme concentration in RA SF than in the SF from patients with trauma (stromelysin 40.1 +/- 26 micrograms/ml [mean +/- SD] in RA SF, 8.5 +/- 15 micrograms/ml in trauma SF; collagenase 2.2 +/- 3.3 micrograms/ml in RA SF, 1.1 +/- 2.3 micrograms/ml in trauma SF). The majority of the stromelysin within the SF bound to reactive red-agarose and was identified as prostromelysin based on electrophoretic mobility and immunoblotting with monospecific antibodies. CONCLUSION: The finding of high levels of stromelysin in SF from patients with RA supports the proposal that this enzyme may play a role in the connective tissue degradation observed in this disease.

Correlation in the expression of type IV collagenase and the invasive and chemotactic abilities of myelomonocytic cells during differentiation into macrophages.

Monomyelocytic phagocytes originate in the bone marrow and while differentiating into macrophages migrate to inflammatory foci and target tissues by egress from the capillary blood vessels. During such diapedesis, the cells must traverse tissue barriers such as basement membrane, which has type IV collagen as its principal structural element. We studied whether the expression of type IV collagenase activity, invasion through basement membrane, and the response to inflammatory chemoattractants are related to each other and to the process of differentiation of murine M1 myeloid leukemia cells into macrophages. M1 cells stimulated with mouse lung-conditioned medium (MLCM) or interleukin 6 (IL6) differentiate into macrophages by 72 h, as determined by expression of Fc receptors, induction of lysozyme, and morphological changes from blast cells to mature macrophages. During this process of differentiation the invasive ability of the cells and the amount of type IV collagenase in the supernatants from the invading cells continuously increased up to 72 h. Zymographic analysis of supernatants of the invading cells revealed a single 100-kd metalloproteinase with gelatinolytic activity. Chemotaxis towards arachidonic acid metabolites, which are present in inflamed tissues, was detected only in differentiated cells. Studies with thioglycolate (TG)-elicited peritoneal macrophages gave results similar to those obtained with differentiated M1 cells, showing that the ability to invade basement membrane, the expression of type IV collagenase, and the chemotactic response to inflammatory chemoattractants all increased with the differentiation of myeloid cells and reached their highest expression in fully differentiated cells.

Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn.

The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.

An immunoassay for qualitative estimation of collagenase activity in mammalian tissues.

Collagen, the most abundant protein of the mammalian body, is specifically degraded by collagenase. Collagenase activity and subsequent collagen degradation are the main aspects of essential biological processes such as bone remodeling, tissue repair and wound healing. Measurement of collagenase activity is of interest to a wide variety of investigations using mammalian tissues, including clinical specimens. Most assays for collagenase activity are based on chemical modification of the substrate collagen. A unique feature of the present method is that it allows the rapid, qualitative measurement of collagenase activity without the requirement of substrate modification. It is based upon both substrate digestion by collagenase and reaction of undigested collagen with its antibody. Collagenase activity is measured by quantitation of immunoreactivity of undigested collagen using an enzyme-linked immunosorbent assay (ELISA). The assay is performed in 96-well microtiter plates used for ELISA. The advantages of this method are several: (a) a highly specific reaction between substrate collagen and its antibody that rules out the possibility of nonspecific quantitation; (b) the use of a nonmodified substrate; (c) the ease and rapidity of performance of a microassay. Application of the microassay to mammalian tissue homogenates was demonstrated in rat uterus tissue and ventricular myocardium of normal and hypertensive rats.