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This review summarizes the current state of knowledge regarding the proteins composing the extracellular matrix in the human prostate. The normal expression as well as the changes which occur in PIN and carcinoma are described for the lamins, collagens, and glycosaminoglycans.
Deer antler regeneration is a uniquely intense and complex process, which involves chondrogenic and intramembranous ossification. Cell differentiation in the developing antler of red deer, Cervus elaphus, was characterized with extracellular matrix markers. Expression of the four matrilin genes was monitored by immunohistochemistry and in situ hybridization and compared to cartilage markers collagen II and cartilage link protein, the bone component collagen I, and the endothelial basement membrane constituent laminin. The mesenchyme layer at the very tip of the velvet antler was enriched in link protein, indicative of the role of hyaluronan in apical morphogenesis. Matrilin-2, formerly described as a component of hard and soft connective tissue matrices, was identified here also as a marker of cells with high differentiation potential: it is expressed predominantly by mesenchyme cells, prechondrocytes and preosteoblasts. In addition to matrilin-3, documented as a component of the bony extracellular matrix, expression of the other three matrilin genes was observed in osteoprogenitor cells and osteoblasts. A layer of presumed osteoprogenitor cells, which surrounded the perivascular channels, expressed all four matrilins and collagen I. As a consequence, all four matrilins, including matrilin-1, previously detected in the skeleton only in cartilage, were found associated to collagen I-rich structures in a thin layer bordering the columns of hypertrophic chondrocytes. Cells with similar morphology and expression pattern were identified in the periosteum. Altogether all cell types of the chondrogenic and osteogenic lineage that expressed the four matrilins were in a separate study [Faucheux, C., Nicholls, B.M., Allen, S., Danks, J.A, Horton, M.A., Price, J.S., 2004. Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler. Dev. Dyn. 231, 88-97] positive for parathyroid hormone-related peptide and its receptor.
Alterations in the composition of intervertebral disc extracellular matrix, mainly collagen and proteoglycans, may cause changes in mechanical properties of the disc, leading to dysfunction, nerve root compression, and herniation with severe clinical manifestations. Matrix metalloproteinases may be involved in degradation by hydrolysing extracellular matrix components. Inhibitors of matrix metalloproteinases, in contrast, function in the maintenance of degradation control. In this study, we investigated: (i) whether the level of matrix degradation correlated with the duration of the symptomatic disease, (ii) roles of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) in intervertebral disc degeneration. Nucleus pulposus of intervertebral discs were obtained from 22 patients and analysed for collagen and proteoglycan contents, and pro-MMP-2, TIMP-2 levels. Collagen content was determined as hydroxyproline and proteoglycan content was measured as glycosaminoglycans. The loss in matrix components did not correlate with the duration of the degenerative disc disease. Pro-MMP-2 levels were higher at early stages of the degenerative disc disease (r = -0.495, P < 0.05). TIMP-2 levels were similar in all samples. Pro-MMP-2 and TIMP-2 levels negatively correlated in herniated discs samples (r = -0.855, P < 0.01). Pro- MMP-2 levels negatively correlated with the collagen content in herniated disc material. Our findings may suggest a silent period of active disease prior to symptomatic outcome during which irreversible matrix loss occurs. Involvement of other proteolytic enzymes at different stages of the disease should also be investigated to help to control the degradation cascade at relatively early stages of disc degeneration before the clinical onset of disease.
It have been reported that chronic drug abuse induces changes in extracellular matrix (ECM) composition of myocardium. Matrix metaloproteinases (MMP) play important role in ECM remodelling and alteration in the ECM of myocardium. They are able to cleave ECM components and may contribute to progression of heart damage. Remodelling implies an alteration in the ECM and in the spatial orientation of cells and intercellular components. In this report we evaluated the extracellular matrix of myocardium of deceased drug abusers and controls. The myocardium of 5 toxicologically positive cases and 4 control cases were analysed by zymography of MMP, electrophoresis of collagens and histologicaly. Gelatinases (MMP-2 and MMP-9) and collagenase (MMP-1) activities were increased in drug abusers compare to the controls. In contrast, alpha chain of collagen type I after 160 hours collagen extraction, were not observed in control specimens. The activation of MMP proenzymes is one of the critical steps that lead to ECM breakdown. Our findings suggest that the myocardial collagen matrix is damaged in drug abusers.
We have previously shown that G8-1, a murine clonal skeletal-muscle cell line, produces a substrate-attached extracellular matrix [Beach, Burton, Hendricks & Festoff (1982) J. Biol. Chem. 257, 11437-11442]. To examine further the expression of extracellular-matrix proteins by muscle cells, we have analysed the collagenous proteins secreted by G8-1 myoblasts. We have found that collagens and/or procollagens, corresponding to genetic types I, III and IV (and possibly V), are produced and secreted by G8-1 myoblasts. The major secreted collagenous polypeptides were identified as alpha 1 type I and its precursors by using pulse-chase studies, pepsin and collagenase digestions and CNBr fragmentation. The presence of lesser amounts of the other collagens was determined by immunoprecipitation. These results demonstrate that clonal skeletal-muscle cells, in the absence of fibroblasts and an exogenous collagen substrate, are able to synthesize and secrete several extracellular-matrix collagenous proteins in proportions similar to those which are commonly found in muscle tissue and mixed cultures of muscle cells and fibroblasts.
Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.
We studied the synthesis, secretion, and aggregation into the extracellular matrix of fibrillin by dermal fibroblasts from 26 probands with the Marfan syndrome. Cells from seven probands synthesized approximately half the normal amount of fibrillin when compared with intrafamilial or unrelated controls. Cells from an additional seven probands synthesized a normal amount of fibrillin but secreted the protein less efficiently than control cells. Cells from a further eight probands synthesized and secreted normal amounts of fibrillin but the protein was poorly incorporated into extracellular matrix. Cells from the remaining four probands were indistinguishable from control cells in their synthesis and processing of fibrillin. Cells from 18 family members of 10 of the probands were also studied. Cells from affected individuals in the same family had the same biochemical defect and those from unaffected family members were indistinguishable from controls. These results indicate that mutations in the gene that encodes fibrillin are responsible for the Marfan syndrome in the majority of individuals (confirming recent immunohistochemical and genetic linkage studies) and that a variety of mutations can produce the phenotype associated with the syndrome.
Cumulus oophorus, an investing structure unique to oocytes of higher mammals, is induced to synthesize an extensive extracellular matrix by ovulatory stimulus, leading to the characteristic preovulatory expansion of the cumulus-oocyte complex. The extracellular matrix consists of cumulus cell-secreted hyaluronan, proteoglycans and proteins, as well as extrafollicularly originated SHAPs (serum-derived hyaluronan-associated proteins) that are bound covalently to hyaluronan. The secretion and assembly of matrix molecules by cumulus cells are temporally regulated by factors derived from both mural granulosa cells and oocyte, which synchronize the deposition of the cumulus oophorus matrix with other intrafollicular ovulatory events. The cumulus oophorus matrix is essential for ovulation and subsequent fertilization. Recently, taking advantage of animal models with defined genetic modifications, it has become possible to investigate in vivo the structure of the cumulus oophorus matrix, the regulatory mechanism for matrix deposition and its biological functions. This review focuses on the recent findings on the construction of the cumulus oophorus matrix and the regulation.
Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.
A variety of cell types at the blastocyst implantation site produce growth factors that could play important role(s) in the implantation process. Recent evidence indicates that decidual cells and/or embryos produce transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and colony-stimulating factor (CSF-1). Furthermore, receptors for EGF, PDGF, and CSF-1 have been detected on embryonic and trophoblastic cells. The purpose of this study was to investigate the effects of these growth factors and possibly growth factor-extracellular matrix interactions on mouse blastocyst attachment and trophoblast outgrowth in vitro. Various dilutions of the growth factors TGF-alpha, EGF, PDGF, FGF, and CSF-1 were added to cultures of 5-day-old hatched blastocysts in fibronectin-coated plastic wells. Blastocysts were scored for attachment, trophoblast outgrowth, and surface area at 24, 48, and 72 h. Each of these growth factors significantly enhanced trophoblast outgrowth, and a cocktail containing all of the growth factors had a significantly stronger effect. PDGF and FGF, but no other growth factors, also enhanced trophoblast outgrowth following pulsatile incubation with the fibronectin matrix coating of the culture wells, indicating that interactions between these growth factors and extracellular matrix elements could influence implantation. This study suggests that various growth factors may play an important role in the implantation process, that synergistic effects may be obtained by combinations of growth factors, and that interactions between certain growth factors and extracellular matrix elements may be significant.(ABSTRACT TRUNCATED AT 250 WORDS)
Human kallikrein 4 (hK4) is a member of the expanded family of human kallikreins, a group of 15 secreted proteases. While this protein has been associated with ovarian and prostate cancer prognosis, only limited functional information exists. Therefore, we have undertaken an investigation of its enzymatic properties regarding substrate preference, degradation of extracellular matrix proteins, and its inhibition by various inhibitors. We successfully expressed and purified active recombinant hK4 from supernatants of the Pichia pastoris expression system. This enzyme seems to cleave more efficiently after Arg compared to Lys at the P1 position and exhibits modest specificity for amino acids at positions P2 and P3. hK4 forms complexes with alpha1-antitrypsin, alpha2-antiplasmin and alpha2-macroglobulin. The protease mediates limited degradation of extracellular matrix proteins such as collagen I and IV, and more efficient degradation of the alpha-chain of fibrinogen. The cleavage of extracellular matrix proteins by hK4 suggests that this enzyme may play a role in tissue remodeling and cancer metastasis.
OBJECTIVE: To study the effect of extracellular matrix of xenogenic femoral fascia in repair of renal trauma. METHODS: Twelve adult dogs were used and randomly assigned to 6 groups, and the animals were sacrificed separately in 1 and 2 weeks 1,2,4 and 8 months after renal repair operations. The examinations of blood and urine routine, blood urea nitrogen and creatinine, electrolyte and serum renin were performed before and after operations at various times. The creatinine clearances of affected and contralateral normal kidneys were evaluated before death and the local areas of renal repair were studied by light and electron microscopy. RESULTS: Bleeding was stopped completely after the entire patch was sutured, and only mild adhesions to around tissues were found in various times after operations. As time passed, the repair patch was replaced by smooth neocapsule like normal renal capsule. CONCLUSION: Extracellular matrix of xenogenic femoral fascia might be an ideal tissue engineering material for renal repair.
OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators.
Tumor cells interact with stromal cells via soluble or cell-bound factors stimulating the production of matrix metalloproteinases (MMPs), a group of enzymes largely involved in the extracellular matrix (ECM) remodeling in tumor invasion. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate in vitro the fibroblast production of various MMPs such as interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2). In this study, the EMMPRIN protein was detected by immunohistochemistry prominently in malignant proliferations of the breast and the lung. It was present at the surface of both tumor epithelial and peritumor stromal cells. Because previous studies have reported that stromal cells do not express EMMPRIN mRNAs, it is very likely that EMMPRIN is bound to stromal cells via a specific receptor. Moreover, our observations also demonstrated that the same peritumor stromal cells strongly express MMP-2. Our results show that EMMPRIN is an important factor in tumor progression by causing tumor-associated stromal cells to increase their MMP-2 production, thus facilitating tumor invasion and neoangiogenesis. (J Histochem Cytochem 47: 1575-1580, 1999)
UNLABELLED: In spite of progress in biotechnology, isolation of porcine pancreatic islets remains a difficult task with unpredictable results. One reason could be the lack of knowledge as to the expression of extracellular matrix proteins in porcine exocrine and endocrine tissues, particularly in "islet capsules". Such proteins are subject to digestion by proteases, yet they might have a protective function for the fragile islets. OBJECTIVE: Of our study was a detailed histological analysis of the extracellular matrix proteins in various pig breeds. A broad panel of commercial, human-specific antibodies were used, since antibodies against porcine tissue were not available. METHODS: Frozen pancreatic tissue section of 7 domestic pig breeds, the Goettingen Minipig and the Wild Boar were stained with antibodies against collagen types I, II, III, IV, V, VI, VII, IX, laminin, fibronectin, vitronectin and elastin. Binding of antibodies was detected by immunoperoxidase and evaluated microscopically. Human and rat tissue was treated in the same way. RESULTS: (1) With the exception of anti-collagen type II, type VII and vitronectin, all antibodies revealed distinct binding patterns in the pancreas of the different pig breeds. However these antibodies bound on human cartilage and skin. (2) Collagen types I, III, IV, laminin and fibronectin are expressed on porcine pancreatic "islet capsules". (3) Expression levels of these proteins on "islet capsules" vary in the different pig breeds. However, no significant differences could be found in the expression pattern of collagen types I, III, IV, laminin and fibronectin, comparing domestic, experimental and wild type pigs. (4) Older individuals (Goettingen Minipig) appear to express higher levels of proteins on "islet capsules" than younger ones. CONCLUSIONS: Antibodies with specificity for human extracellular matrix proteins can be used successfully in the porcine pancreas. Thus, analysis of the structure and composition of porcine pancreatic tissue can be performed even without pig-specific antibodies. Particularly, the effects of various proteases and collagenases on the pancreatic tissue can now be monitored by immunohistochemical analysis allowing a rational design of protease mixtures for the isolation of pancreatic islets.
Acidic and basic fibroblast growth factors are heparin-binding proteins that induce cellular proliferation, mesodermal development, and vascular growth. As such, they may be important in cardiac development and disease. To determine whether cardiac myocytes contain fibroblast growth factors, neonatal rat cardiac myocytes were studied in primary culture and compared to primary cultures of nonmyocyte cardiac cells. Northern blot analysis revealed a 4.0-kilobase mRNA in myocytes that hybridized to acidic fibroblast growth factor cDNA and was not detectable in nonmyocyte cultures. Western blot analysis demonstrated the accumulation of a 15-kDa peptide with immunological identity to acidic fibroblast growth factor in extracts of extracellular matrix from myocyte cultures that was not detectable in similar extracts of nonmyocyte extracellular matrix. No acidic fibroblast growth factor-like protein was detectable in cellular lysates from either myocyte or nonmyocyte cultures. These results demonstrate that neonatal cardiac myocytes express acidic fibroblast growth factor mRNA and deposit a protein with immunological identity to acidic fibroblast growth factor into the extracellular matrix. The results suggest that acidic fibroblast growth factor produced by cardiac myocytes may mediate, through both paracrine and autocrine mechanisms, such diverse processes as myocyte differentiation, cellular proliferation, and vascular growth in the heart.
A two-chamber assembly has been employed to investigate influences of peritubular cells (PC) and extracellular matrix (Matrigel) on barrier formation by Sertoli cells (SC) in culture and on transferrin production. The kinetics of transport of [3H]inulin across a Millipore filter were essentially the same in the presence or absence of Matrigel or PC. In contrast, a SC monolayer retarded the diffusion of [3H]inulin, increasing the estimated time for 50% equilibration from about 4 h to approximately 12 h. Matrigel and PC each independently further increased the equilibration time, with the largest effects elicited by the presence of Matrigel (approximately 21 h). Data have been interpreted to indicate that these two components, especially extracellular matrix, facilitate the formation of a functional barrier by SC in the two-chamber system. PC assume a more important role than Matrigel in the modulation of transferrin secretion by SC. Transferrin concentrations were higher in the inner chamber, corresponding to those in the adluminal compartment, but transferrin masses were higher in the outer chamber under the conditions described. We report the effects of the presence and absence of Matrigel, PC, and FSH on levels of transferrin secreted by SC. Addition of FSH resulted in increased transferrin secretion by SC maintained under all conditions examined. We compare our data with those previously reported by others and attempt to provide a basis for the differences observed. We discuss the properties of the system and outline major advantages and limits of the two-chamber assembly in investigations on the polarity and properties of SC in culture.
The distribution of elastin-like immunoreactivity around small lymphatic vessels was investigated in three different human tissues (skin, heart and dental pulp) using high resolution immunocytochemistry. Quantitative assessment of the immunogold reaction was performed with an image analysis system. Intense and moderate elastin-immuno-reactivity was detected in the extracellular matrix around small lymphatic vessels of the skin and heart, respectively. By contrast, absence of immunostaining was observed around lymphatic vessels in the dental pulp. Although the staining was mostly detectable on the non-fibrillar amorphous component of the extracellular matrix, some microfibrils were also immunostained in close proximity to the lymphatic vessel wall. These findings support the concept that small lymphatic vessels may be heterogeneous with respect to the composition of the extracellular matrix around their wall. The observation that it is possible to observe small lymphatic vessels displaying low or no elastin-immunoreactivity in the adjoining matrix militates against the hypothesis that elastic fibers play a pivotal role in the mechanisms that regulate the function of small lymphatic vessels.