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PubMed1991

Rod and cone specific domains in the interphotoreceptor matrix.

The insoluble matrix domain of the interphotoreceptor matrix (IPM) from normal dog, cat, and mouse retinae were characterized using lectin cytochemistry. The lectins WGA (wheat germ agglutinin) and PNA (peanut agglutinin) were used to label interphotoreceptor matrix microdomains in cryosections of retinal tissue and in extracted insoluble matrix. Retinal cryosections and extracted matrix were examined by epifluorescence microscopy and scanning confocal laser microscopy, the latter allowed for the removal of all background fluorescence and gave increased resolution. The insoluble matrix was extracted as a continuous sheet that was comprised of two photoreceptor-specific matrix domains distinguished both by the size of the domains, and by differential binding of WGA and PNA lectins. Each domain encloses a photoreceptor inner and outer segment. Individual rod-associated domains were connected into a hexagonal lattice and this pattern was regularly interrupted by the larger cone-associated domains which have 8-10 surrounding rod domains. The PNA lectin primarily labeled the cone-associated matrix with faint binding to the rod matrix; the WGA lectin labeled both the rod- and cone-associated matrix.

Animals↗
PubMed1984

The synthesis of subendothelial matrix by bovine aortic endothelial cells in culture.

Bovine aortic endothelial cells cultured on collagenous or plastic substrata continuously synthesize and deposit a subendothelial matrix, independently of whether the cells are in the logarithmic or the stationary phase of growth. This subendothelial matrix contains fibrillar and amorphous elements comparable with those observed in the subendothelium in vivo. Deposition of subendothelial matrix on a collagen gel substratum both started earlier and progressed at approximately double the rate than that on denatured collagen. The relative composition of the subendothelial matrix was assessed by sequential incubation with trypsin, elastase and collagenase (Jones et al., 1979). The subendothelial matrix deposited on collagen gels by early confluent cultures and late post-confluent cultures differed in their enzyme sensitivity. These age-related changes in the enzyme sensitivity of the subendothelial matrix were characteristic for each cloned cell population examined. Comparable variations in the composition of the subendothelial matrix were not observed when the cells were cultured on plastic or gelatin-coated dishes; the subendothelial matrix deposited on these two substrata contained considerably more trypsin-sensitive material and less elastase and collagenase-sensitive material than the matrix deposited on native collagen gels. Age-related changes in the enzyme sensitivity of the subendothelial matrix deposited on collagen gels was found to be a function of the time elapsed since confluence and it was not related to the time elapsed since plating or to the number of cells present.

Animals↗
PubMed1984

Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts.

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin -producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.

Age Factors↗
PubMed1992

Immunohistochemical localization of matrix proteins in the femoral joint cartilage of growing commercial pigs.

The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular-epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycan was more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Septal distribution and the relationship of matrix vesicle size to cartilage mineralization.

To estimate matrix vesicle distribution between longitudinal and transversal septal matrix in the proliferative, hypertrophic and calcifying zones of normal epiphyseal cartilage, the volume density of matrix vesicles in the longitudinal septal matrix was compared to that of total extralacunar matrix. The results confirm the qualitative observation by Anderson that matrix vesicles are located mainly in the longitudinal septa. To elucidate whether cartilage mineralization can be related to the disappearance of matrix vesicles of particular size classes, epiphyseal growth cartilage from three groups of animals were studied: normal rats, rats with florid rickets and rats with early healing rickets. The study was focused on the proliferative, hypertrophic and calcifying zones and in each zone the matrix vesicles were classified into four size classes: 1, less than or equal to 50 nm; 2, 51-67 nm; 3, 68-84 nm; 4, greater than or equal to 85 nm. The results show that the decrease in volume density previously demonstrated in normal rats to a large extent is due to a decreased number of larger vesicles. In florid rickets the decrease in this size group is much smaller while the values for healing rachitic animals fall between those of florid rachitic rats and those of controls. The data indicate that the decrease in the number of larger vesicles, which represents a considerable vesicle volume, is of particular importance. The heterogenous change in the concentration of matrix vesicles of different size classes during cartilage mineralization as well as under conditions of arrested calcification, is compatible with the existence of a matrix vesicle subpopulation of larger size.

Animals↗
PubMed1986

Mesangial cell hillocks. Nodular foci of exaggerated growth of cells and matrix in prolonged culture.

To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation of matrix similar to that seen in the mesangium of diseased glomeruli. It is concluded that the in vitro model of prolonged MC outgrowth may facilitate the investigation of factors that govern mesangial matrix production. Such a model could be used in examining the response of the mesangium to defined inflammatory or metabolic stimuli.

Animals↗
PubMed1987

Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins.

We devised a model system to study the effects of extracellular matrix proteins on the malignant phenotype of an anaplastic glioma cell line, U 343 MG-A. Well-characterized cultures derived from normal human leptomeninges were grown to confluence and maintained for 2 weeks. The leptomeningeal cells were then removed with base and detergent, leaving behind an extracellular matrix enriched in laminin, fibronectin, type I and IV collagen, and procollagen III. U 343 MG-A tumor cells planted on top of this normal extracellular matrix were profoundly growth inhibited compared with glioma cells grown on plastic alone. Glioma cells grown on the extracellular matrix developed multiple, slender processes and assumed a more differentiated astrocytic phenotype; immunostains for glial fibrillary acidic protein revealed a more extensive intracytoplasmic network of intensely staining filaments than in control glioma cells. When glioma cells grown on the extracellular matrix were analyzed by an enzyme-linked immunosorbent assay for glial fibrillary acidic protein, the amount of this intermediate filament per cell was increased 20-fold compared with glioma cells growing on plastic. The growth and differentiation of U 343 MG-A glioma cells in flasks coated with purified fibronectin or laminin was not significantly perturbed; however, glioma cell cultures grown in flasks coated with purified type I or IV collagen showed decreased cellular proliferation, stellate cell formation, and increased levels of glial fibrillary acidic protein per cell compared with glioma cells growing on plastic. Gelatin gel analysis showed that U 343 MG-A glioma cells growing on plastic secreted a 65,000-D metalloproteinase that was not secreted by glioma cells grown on the leptomeningeal extracellular matrix. We conclude that in this system, the extracellular matrix of a normal human leptomeningeal culture substantially inhibited the proliferation of and induced differentiation in an anaplastic glioma cell line. Our analysis of single components of the extracellular matrix suggests that these effects may be mediated in part by type I and IV collagen. The mechanism by which the leptomeningeal extracellular matrix inhibits glioma cell proliferation may be by diminishing tumor-associated protease secretion so that the degradation of extracellular matrix macromolecules in the tumor cell microenvironment is prevented and tumor cell migration becomes less likely.

Cell Differentiation↗
PubMed1986

Invasion of reconstituted basement membrane matrix by metastatic human tumor cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the invasive properties of human HT1080 fibrosarcoma cells. Within several hours after seeding, the tumor cells initiated a random migration, leaving behind channels etched in the surface of the matrix. Eventually the channels became interconnected into a complex network. As the tumor cells proliferated, the channels became filled until the surrounding matrix was gradually dissolved. Cells then migrated outward, forming the typical disorganized cell monolayer normally observed when fibrosarcoma cells are cultured on plastic surfaces. In contrast to the fibrosarcoma cells, normal skin fibroblasts, while able to attach to the matrix, exhibited minimal migration, tracking, and invasion during the same time period. When tumor cells were seeded onto thick layers of matrix, the cells ultimately invaded downward into the matrix, leaving behind open tunnels. At the front of the invading cells, long irregular pseudopodia projected in the direction of movement. Electron microscopy demonstrated these filopodial and lamellopodial projections to directly extend into the surrounding matrix, with focal clearing of the matrix in the immediate vicinity of these invading pseudopodia. Thus, tumor cell invasion of extracellular matrices, including basal lamina, may proceed by the formation of specialized pseudopodia that not only form adhesion contacts with the matrix but also provide an efficient mechanism for the focal hydrolysis of the matrix at the site of directed cell movement.

Basement Membrane↗
PubMed1983

Role of plasminogen in matrix breakdown by neoplastic cells.

Destruction of the extracellular matrix is often observed during tumor invasion, and proteolytic enzymes may participate actively in the degradation of matrix proteins. The present report elucidates the role of plasminogen in the degradation by tumor cells of an in vitro elaborated extracellular matrix. Matrices produced by rat smooth muscle cells in the presence of [3H]proline or [3H]fucose were used as substrates for human fibrosarcoma cells (HT-1080), mouse melanoma cells (B16F1), or human rhabdomyosarcoma cells (RD). All three cell lines degraded part of the glycoprotein compartment of the matrix. HT-1080 cells digested the matrices in a density-dependent manner, and while matrix glycoprotein degradation was plasminogen-dependent at the beginning of the experiment and at low cell densities, the zymogen was not essential for further glycoprotein digestion at high cell densities. Depletion of plasminogen from the growth medium resulted in a threefold reduction of matrix degradation by B16F1 cells showing a distinct plasminogen dependency at low cell numbers. RD cells digested only matrix glycoproteins, and this degradation was completely dependent on the presence of plasminogen at all cell densities. These results suggested that plasmin generated from plasminogen by a tumor cell-associated plasminogen activator may be most important for matrix hydrolysis at low cell densities, and while certain tumor cell lines showed a definite plasminogen-independent matrix degradation with increased cell numbers, other neoplastic cells hydrolyzed the matrix only in the presence of the zymogen at all cell densities.

Animals↗
PubMed1983

Alkylating agent interactions with the nuclear matrix.

The interrelationship of DNA to the nuclear matrix is integral to the organization of chromatin within the nucleus and to the DNA replication process. The influence of nitrosourea and nitrogen mustard interactions with the nuclear matrix were studied in log phase HeLa cells. Alkylation of the nuclear matrix by chlorozotocin (CLZ) or 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (CCNU) was 1.58 and 1.27 pmoles drug/micrograms protein, respectively, whereas carbamoylation by CCNU was 32.5 pmoles/micrograms. These constituted approximately 30% of the total (nuclear) drug modifications. The structural matricin fibrillar components of the matrix were alkylated and carbamoylated twice as much as the ribonuclear protein elements (RNP). However, when alkylations are measured per microgram of protein, the ratio of covalently bound drug to RNP:matricin was 1.2 for both CLZ and CCNU. The RNP:matricin carbamoylation ratio for CCNU was 0.9. The importance of DNA and matrix protein alkylations to the process of reassociation was studied. Under control conditions, in vitro, approximately 80% of the DNA was associated with the matrix at a protein:DNA ratio (micrograms for micrograms) of 50:1. Direct alkylation or carbamoylation of the matrix proteins did not affect these DNA-protein interactions. However, using in vitro alkylated DNA (1 alkylation/10(2) base pairs), there was a 60% reduction of the alkylated nucleic acid bound to the matrix at the same protein: DNA ratio. The reduced binding of DNA to matrix may be a function of interference with the DNA recognition sites by alkylation of specific bases. The interference of DNA-matrix association by DNA alkylation may contribute to the cytotoxic activity of these antineoplastic agents.

Alkylating Agents↗
PubMed1988

Noise control in solid-phase immunoassays by use of a matrix coat.

This paper describes the matrix coat noise control technique as applied in an ELISA to detect IgG antibody to the semen protein p30 (also known as prostate-specific antigen). Background noise due to non-specific binding of IgG in solid-phase immunoassays has recently been shown to be highly charge dependent. In this technique, the surface antigen (here, p30) is co-coated on the test surface with an anionic macromolecule, the noise reduction matrix component, to reduce non-specific IgG binding. A second matrix component, the noise balancing component, is added if necessary to balance non-specific IgG binding between a detecting well which contains matrix plus antigen and a control well which contains matrix alone. Sample noise can then be measured in the control well and subtracted to yield a noise-corrected signal. This approach both minimizes background noise and then precisely quantitates residual noise. In this study, human alpha 1-acid glycoprotein (AGP) served as the noise reduction component and bovine serum albumin (BSA) as the noise balancing component. Optimal coating concentrations of AGP and BSA for the matrix were determined by a new technique, the tetrad method of signal and noise analysis. A signal probe (immune serum), a noise probe (non-immune serum) and a set of four test wells are employed to analyze the noise control properties of a given combination of matrix components. For each such tetrad of wells, three ratios are calculated: the sensitivity, signal-to-noise and noise balance ratios, along with a composite index, the matrix index. These can be used to select the combination and concentrations of matrix components which optimize assay noise control and minimize losses of assay sensitivity. When an ELISA using matrix coat noise control (MCNC-ELISA) was compared with ELISAs using standard blocking techniques, the MCNC-ELISA was superior in its ability to control for false positive results due to non-specific IgG binding and binding of the polycation poly-L-lysine. Two subjects (14%) out of a clinic population of sexually active women were found to have significant serum levels of anti-p30 IgG (P less than or equal to 0.001) by MCNC-ELISA.

Antigens, Neoplasm↗
PubMed1983

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation.

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17beta-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or epsilon-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0

Animals↗
PubMed1991

Enhanced assembly of basement membrane matrix by endodermal cells in response to fibronectin substrata.

Basement membranes are complex extracellular matrices contributing to the regulation of growth, migration and differentiation of many cell types. However, little is known about the mechanisms regulating the deposition and assembly of basement membrane from its constituents. We have investigated the role of extracellular matrix molecules in the control of basement membrane matrix assembly by cultured endodermal (PFHR-9) cells. In the presence of fibronectin-depleted serum, substrata of fibronectin or laminin induced an increase in deposition of laminin, type IV collagen and proteoglycans by PFHR-9 cells, in comparison to cells adherent to type I collagen-coated, vitronectin-coated or uncoated substrata. Direct effects of fibronectin or laminin on the degree of cell spreading or rate of proliferation were not responsible for enhanced matrix deposition. The effect did not result from a redirection of basement membrane components to the matrix, since there was no decrease in matrix constituents released to the culture supernatants. Furthermore, the synthesis and release of other molecules that are not basement membrane constituents was unaltered in response to different extracellular matrix substrata. Experiments with fibronectin fragments showed that a 105 x 10(3) Mr 'cell'-binding domain (containing the cell attachment sequence Arg-Gly-Asp-Ser) was an important contributor to enhanced matrix deposition, while the N-terminal 29 x 10(3) Mr heparin-binding domain also contributed to the effect, particularly with respect to heparan sulfate proteoglycan deposition. It seems that fibronectin has a dual role of action in promoting basement membrane matrix assembly, through direct cell surface interactions, and through the binding of fibronectin to other matrix components that may nucleate or stabilize the matrix assembly.

Amino Acid Sequence↗
PubMed1986

Preferential binding of DNA primase to the nuclear matrix in HeLa cells.

Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.

Adenosine Triphosphate↗
PubMed1990

Deposition of extracellular matrix along the pathways of migrating fibroblasts.

Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.

Animals↗
PubMed1992

In vivo interactions of TGF-beta and extracellular matrix.

TGF-beta, a multifunctional cytokine, plays an important role in embryogenesis and in regulating repair and remodeling following tissue injury. Many of the biological actions of TGF-beta are mediated by widespread effects on deposition of extracellular matrix. TGF-beta stimulates the synthesis of individual matrix components including proteoglycans, collagens and glycoproteins. TGF-beta also blocks matrix degradation by decreasing the synthesis of proteases and increasing the synthesis of protease inhibitors. Finally, TGF-beta increases the synthesis of matrix receptors and alters their relative proportions on the surface of cells in a manner that could facilitate adhesion to matrix. All of these events have largely been demonstrated in vitro in cultured cells. In an experimental model of glomerulonephritis we have shown that TGF-beta is responsible for the accumulation of pathological matrix in the glomeruli following immunological injury. Furthermore, all three of TGF-beta's actions on extracellular matrix--increased synthesis, decreased degradation and modulation of receptors--have now been documented to be involved in matrix deposition in vivo in this model. Administration of the proteoglycan decorin suppressed TGF-beta-induced matrix deposition in the nephritic glomeruli, thus confirming a physiological role for decorin as a regulator of TGF-beta. Inhibitors of TGF-beta may be important future drugs in treating fibrotic diseases caused by overproduction of TGF-beta.

Animals↗
PubMed1989

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Actin Cytoskeleton↗
PubMed1992

The extracellular matrix of cartilage in the growth plate before and during calcification: changes in composition and degradation of type II collagen.

Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extracellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗