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PubMed1998

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Adenosine Triphosphate↗

PubMed1999

Optimum spray congealing conditions for masking the bitter taste of clarithromycin in wax matrix.

The effects of operating conditions in the spray-congealing process on the release and the micromeritic properties of clarithromycin (CAM) wax matrix were evaluated. CAM wax matrix with 30% CAM, 60% glyceryl monostearate (GM) and 10% aminoalkyl methacrylate copolymer E (AMCE) was manufactured at various atomizer wheel speeds and liquid feed rates with a spray dryer. Release of CAM from the matrix exhibited a two-phase pattern, probably due to the dissolution of the fine portions broken on the surface of the matrix. The slope and the extrapolated y-intercept of the subsequent release pattern were defined as the release rate and the initial amount of release of CAM from the matrix, respectively. These release parameters, as well as the volume median diameter and the specific surface area of matrix, were selected as response variables, and multiple regression analysis was performed. For specific surface area and initial amount of release, a minimum point was observed on the contour curve when the atomizer wheel speed was constant and the liquid feed rate was varied. For the release rate, a maximum point was observed on the contour curve under the same conditions. These points were considered preferable for masking the bitter taste of CAM preparation. Microscopic observation revealed that a small spherical matrix with a smooth surface could be obtained with a high atomizer wheel speed and optimum liquid feed rate. This matrix also possessed excellent properties for taste masking, with small initial amount of release and subsequent high rate of release. In conclusion, the congealing speed of melt droplets was the dominant factor in masking the bitter taste of CAM.

Algorithms↗

PubMed2004

Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization.

UNLABELLED: Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. INTRODUCTION: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. MATERIALS AND METHODS: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. RESULTS: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. CONCLUSIONS: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.

Animals↗

PubMed2005

Mathematical modelling and controlled drug delivery: matrix systems.

This paper deals with the physical and mathematical modelling description of drug release from matrix systems. In the introduction, matrix systems are considered in the wide frame of the controlled release systems and the concept of mathematical model is briefly discussed. Then, matrix structure and topology are matched, analysing the characteristics of the three-dimensional network constituting them. In this context, drug release mechanisms are considered with particular emphasis on the key factors ruling the release kinetics, such as matrix swelling, erosion, drug dissolution (re-crystallisation), drug diffusion, drug - polymer interaction, initial drug distribution and particle size distribution (for powdered matrix systems). The mathematical modelling section firstly considers the empirical and semi-empirical models that have the great advantage of showing analytical solutions. Then, the attention is focused on theoretical approaches regarding matrix swelling equilibrium and kinetics, drug dissolution, drug diffusion, drug - polymer interaction, initial drug distribution and matrix erosion. Finally, release kinetics from polydispersed spherical particles is studied. This review points out the fact that the comprehension of the phenomena ruling drug release from matrix systems is appropriate from both the physical and modelling point of view, although further improvements are always possible and desirable.

Algorithms↗

PubMed2005

In vitro and in vivo evaluation of guar gum-based matrix tablets of rofecoxib for colonic drug delivery.

The present study was carried out to develop and evaluate guar gum-based matrix tablets of rofecoxib for their intended use in the chemoprevention of colorectal cancer. Matrix tablets containing 40% (RXL-40), 50% (RXL-50), 60% (RXL-60) or 70% (RXL-70) of guar gum were prepared by wet granulation technique, and were subjected to in vitro drug release studies. Guar gum matrix tablets released only 5 to 12% of rofecoxib in the physiological environment of stomach and small intestine. The matrix tablets RXL-40 disintegrated completely within 10 h in a dissolution medium without rat caecal contents (control study), and hence not studied further. When the dissolution study was continued in simulated colonic fluids (rat caecal content medium), the matrix tablets RXL-50 were acted upon by colonic bacterial enzymes releasing the entire quantity of drug wherein there was no appreciable difference when compared to that released in control study. The matrix tablets RXL-60 released another 88% of rofecoxib whereas matrix tablets RXL-70 released only 57% of rofecoxib in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents. The guar gum matrix tablets RXL-70 were subjected to in vivo evaluation in human volunteers to find their ability of targeting rofecoxib to colon. The delayed Tmax, prolonged absorption time (ta), decreased Cmax and decreased ka indicated that rofecoxib was not released significantly in stomach and small intestine, but was delivered to colon resulting in a slow absorption of the drug and making it available for local action in human colon.

Adolescent↗

PubMed2001

Time dependent smooth muscle regeneration and maturation in a bladder acellular matrix graft: histological studies and in vivo functional evaluation.

PURPOSE: We evaluated the time dependence of smooth muscle regeneration and restoration of in vivo functional properties in bladder augmented with a bladder acellular matrix graft. MATERIALS AND METHODS: A total of 45 Sprague-Dawley rats underwent augmentation cystoplasty with a bladder acellular matrix graft. Two rats each were sacrificed at various intervals within the first 21 days and 6 each were sacrificed at 4, 8 and 12 weeks. This second group underwent preoperative and postoperative assessment of bladder function, including cystometry, electrostimulation and stimulation with ice water, potassium and carbachol, as well as labeling of the bladder wall by the injection of fluorescent microspheres. After sacrifice slides of the bladders prepared for hematoxylin and eosin, trichrome, KI67, vimentin, desmin, smooth muscle specific alpha-actin and fluorescent microspheres were evaluated. RESULTS: Within 2 weeks the number of cells in the matrix as well as the proliferation index increased rapidly and then decreased gradually. Erythrocytes and inflammatory cells were found in the matrix within 2 to 4 days, followed by fibroblasts. A bladder host-to-matrix shift was evident by the appearance of microspheres in the matrix. Cell marker expression indicated the early appearance of vimentin and alpha-actin within the first 10 days. Distinct desmin expression was observed later, when the first smooth muscle cells were recognized. Functional evaluation revealed restored bladder function at 12 weeks. CONCLUSIONS: The time dependent increase of muscle cell markers during smooth muscle cell regeneration in a bladder acellular matrix graft is concordant with the progressive restoration of bladder function. These results may support the bladder acellular matrix graft concept for clinical application.

Actins↗

PubMed2002

Cross-linked matrix tissue sealant protects against mortality and hemorrhage in an acute renal injury model in heparinized rats.

PURPOSE: We evaluated the efficacy of a cross-linked matrix tissue sealant compared with oxidized cellulose and electrocautery in an acute renal injury model. MATERIALS AND METHODS: Sprague-Dawley rats underwent partial nephrectomy after intravenous heparin anticoagulation. The cut surface received 1 of 4 therapies, namely no treatment, cross-linked matrix tissue sealant, electrocautery or Surgicel (Johnson & Johnson, New Brunswick, New Jersey) oxidized cellulose. Blood pressure was continually monitored, survival time was determined and total blood loss was quantified in each animal. RESULTS: Animals that received no treatment, electrocautery or oxidized cellulose died within 13 minutes of injury with a decline in blood pressure to 0 mm. Hg. In cross-linked matrix treated rats hemostasis was immediate, resulting in no blood loss after cross-linked matrix application. Mean arterial pressure +/- SEM was maintained at 136 +/- 6 mm. Hg. All cross-linked matrix treated animals survived. CONCLUSIONS: Cross-linked matrix tissue sealant provided immediate hemostasis and protection from a decline in blood pressure after renal trauma in heparinized rats. Cross-linked matrix tissue sealant may benefit patients with severe renal injuries. In the future cross-linked matrix may be useful for long-term partial nephrectomy repair.

Animals↗

PubMed1976

Analysis of matrix vesicles and their role in the calcification of epiphyseal cartilage.

Extracellular matrix vesicles, which have been shown to be associated with initial calcification of cartilage, were isolated, characterized, and studied with 45calcium isotope to determine whether they could form mineral in vitro. It was found that the isolated matrix vesicles contain a phosphatase, active at neutral pH, which has a very wide specificity and will hydrolyze a variety of nucleotide triphosphates, diphosphates, monophosphates, and other phosphate-containing substrate and metabolites. Acid phosphatase, beta-glucuronidase, and cathepsin D were found to be in the cell fractions, in lysosomes; these enzymes are not present in matrix vesicles and this is additional evidence for the difference between matrix vesicles and lysosomes. Matrix vesicles were found to take up 45Ca even in the presence of low levels of Ca and P1 and also to facilitate precipitation of hydroxylapatite when incubated under physiological conditions in the presence of ATP and other phosphate-containing substrates. Systematic electron probe analysis of a septum of epiphyseal cartilage indicates that matrix vesicles gradually accumulate calcium and then phosphorus and thus facilitate the advance of the calcification front. Adjoinging nonvesicular matrix in the hypertrophic zone, cell cytoplasm, and cell processes had very low levels of calcium and phosphorus in a region where matrix vesicles showed high levels of these elements. New concepts are put forward that take accounts of these findings which provide a better understanding of the sequence of mineralization in growth cartilage.

Alkaline Phosphatase↗

PubMed1992

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.

Acrylic Resins↗

PubMed2004

[Culture of mice marrow stromal stem cells in human acellular cartilage matrix].

OBJECTIVE: To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. METHODS: Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge. The MSCs were embedded in acellular cartilage matrix. After 10 day's combined culture, the specimens were observed with optical and electrical microscope. RESULTS: The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae. CONCLUSION: The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.

Animals↗

PubMed2005

Biocompatibility and recellularization potential of an acellular porcine heart valve matrix.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. The study aim was to investigate the biocompatibility and recellularization potential of an acellular porcine valve matrix. METHODS: Acellular porcine valve matrix contact and extract cytotoxicity was tested against porcine fibroblasts and smooth muscle cells (SMC). Porcine cells were incubated with decellularized aortic valve leaflets and aortic wall, and then assessed for changes in morphology and contact inhibition of growth. Soluble tissue extracts were prepared from decellularized leaflets and aortic wall, and assessed for their effect on the viability of cultured porcine cells. Acellular leaflets were seeded with either fibroblasts or SMC at 1 x 10(3) to 1 x 10(6) cells/cm2 for 24 h, or 5 x 10(4) cells/cm2 for 1-4 weeks. Cell attachment onto, and migration into, the acellular matrix was assessed by scanning electron microscopy and histology. RESULTS: No contact inhibition of growth, or changes in fibroblast or SMC morphology, were observed following contact with the acellular valve matrix. No soluble extract cytotoxicity was found. Intermediate cell-seeding densities (2.5 x 10(4) to 7.5 x 10(4) cells/cm2) of both cell types produced confluent cell attachment; at the lowest concentration (1 x 10(3) cells/cm2) cell attachment was sparse, and at the highest (1 x 10(6) cells/cm2) it was multilayered. The SMC migrated throughout the leaflet matrix over four weeks, but there was no fibroblast migration into the matrix. CONCLUSION: The absence of contact and extract cytotoxicity indicated that the acellular valve matrix was biocompatible in vitro. The failure of porcine fibroblasts to grow on, or infiltrate into, the matrix suggested that the SMC may be the preferred cell type for future leaflet recellularization studies in the development of a tissue-engineered heart valve replacement.

Animals↗

PubMed1990

Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor.

We have previously shown that fibrinogen (fg) acts as a subendothelial matrix protein in promoting human endothelial cell (EC) adhesion and cytoskeletal organization. In this study we report that EC spreading on fg, at variance with other matrix proteins, requires endogenous matrix protein synthesis and secretion. ECs, upon seeding on fg, promptly released and organized a fibronectin (fn) matrix. Fg was more effective than vitronectin (vn) in promoting the deposition of this protein. ECs treated with monensin to block matrix protein secretion still adhered to fg but did not properly organize their cytoskeleton and adhesion structures. In contrast, monensin did not affect EC spreading either on vn or on fn. Using antibodies to the alpha and beta chains of fn (alpha 5 beta 1) and vn (alpha v beta 3) receptors, it was found that ECs adherent to fg show clustering and organization in adhesion structures of both type of receptors. A faint staining of adhesion structures with alpha 2 but not alpha 3 and alpha 6 antibodies was also observed. Antibodies either to vn or fn receptors were able to disrupt the EC monolayer and to induce EC retraction and detachment, thus indicating that both receptors are important in maintaining a sustained EC adhesion to fg. However, when ECs were treated with monensin only the vn receptor was organized in adhesion structures while the fn receptor was diffusely distributed. This suggests that clustering of the fn receptor is mediated by the release of endogenous matrix proteins induced by the exposure to fg. In conclusion, fg has a peculiar and complex type of interaction with ECs since it requires endogenous matrix protein release and the recruitment of more than one adhesive receptor. This suggests a specific way of response of ECs to each extracellular matrix component.

Actins↗

PubMed1990

Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin.

Individual neostriatal-matrix spiny neurons were stained intracellularly with biocytin after intracellular recording in vivo, and their axons were traced into the globus pallidus (GP), entopeduncular nucleus (EP), and/or substantia nigra (SN). The locations of the neurons within the matrix compartment of the neostriatum (NS) were established by immunocytochemical counterstaining of sections containing the cell bodies using antibodies for calbindin-D28K. This allowed nearly complete visualization of the axonal projections of single NS neurons. On the basis of their intrastriatal axonal arborizations, matrix spiny neurons could be divided into 2 types. One type, which was the more common, had local axonal arborizations restricted to the region of the dendritic field, often with axon collaterals arborizing within the dendritic field of the cells of origin. A second, less common, cell type in the matrix had local axon collaterals distributed widely in the NS. Among matrix neurons with restricted local collateral fields, 3 subtypes could be distinguished on the basis of their efferent axonal projections. Type I cells projected only to the GP. Type IIa cells projected to the GP, EP, and SN pars reticulata. Type IIb cells projected to the GP and SN but not to the EP. The shapes and densities of the GP arborizations varied in the 3 cell types, with the cells projecting only to the GP (type I) projecting more heavily and filling a larger volume there than type II cells. The dendrites and intrastriatal axon collaterals of 3 subtypes were similar in morphology. The class of matrix spiny neurons with intrastriatal axon collaterals distributed widely in the NS were observed to project to the GP. Projections beyond the GP were not identified for this cell type, but could not be ruled out. Somatodendritic morphologies of neurons did not differ according to the projection site. These results demonstrate that NS matrix spiny cells are more heterogeneous in their efferent projection patterns than previously suspected on the basis of retrograde axonal tracing and immunocytochemical studies. As predicted by those previous studies, there is a class of matrix neurons that projects only to the GP. Presumably, these cells contain enkephalin. Cells projecting to the SN and EP, and so presumably containing substance P, give off a small projection to the GP, as well, and differ in their collateralization patterns within the 3 major target nuclei.

Animals↗

PubMed1991

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G.

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.

Cartilage↗

PubMed1986

Association of the 3,5,3'-triiodo-L-thyronine nuclear receptor with the nuclear matrix of cultured growth hormone-producing rat pituitary tumor cells (GC cells).

The iodothyronine nuclear receptor, a nonhistone chromatin protein, mediates growth hormone gene transcription in cultured GC cells (Yaffe, B.M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). To determine whether the 3,5,3'-triiodo-L-thyronine (T3) receptor was localized to the nuclear matrix, we studied the subnuclear distribution of [125I]T3-receptor complexes after treatment of nuclei with DNase I and 2 M NaCl to facilitate removal of histones. After incubation with 5 nM [125I]T3 to exchange with 80-90% of nuclear T3 receptors, the nuclear matrix fraction contained less than 1% of nuclear DNA, 16.5% of nuclear protein, and 30-50% (mean, 40.0 +/- 2.3%) of specifically bound [125I]T3. Control experiments showed that nuclear matrix [125I]T3 did not appear exchangeable with added 5 nM T3 and did not result from release and nonspecific precipitation of [125I]T3 or [125I]T3-receptor complexes during nuclear matrix preparation. Studies with the T3 photoaffinity probe, N-2-diazo-3,3,3-trifluoropropionyl-[125I]T3 resulted in the finding of limited capacity receptor forms, 58,000 and 46,000 kDa, in the nuclear matrix. These receptor forms were identical to those observed when N-2-diazo-3,3,3-trifluoropropionyl-[125I]T3-labeled receptors were solubilized directly from nuclei. Lastly, limited capacity [125I]T3 binding was demonstrated in 0.4 M KCl buffer extracts of nuclear matrix. Binding displacement studies suggested that 46% of the binding activity solubilized from nuclear matrix exchanged with [125I]T3 and that the apparent equilibrium association constant of this binding activity was similar to that of 0.4 M KCl extracts of isolated nuclei. These results suggest that an appreciable fraction of nuclear T3 receptors is localized to the nuclear matrix and may influence the expression of thyroid hormone action.

Animals↗

PubMed1983

Degradation of extracellular matrix by larvae of Schistosoma mansoni. I. Degradation by cercariae as a model for initial parasite invasion of host.

We have examined the ability of cercariae of Schistosoma mansoni to degrade a model extracellular connective tissue matrix produced by rat vascular smooth muscle cells in culture. In this model, connective tissue macromolecules are present in the interactive framework that characterizes their structure in vivo. Cercariae were stimulated to degrade the matrix by skin lipid or linoleic acid. At the maximally stimulating concentration of linoleic acid (25 micrograms/cm2), 68% of the total matrix was degraded, including 57% of the glycoprotein, 79% of the elastin, and 8% of the collagen. Degradation of the matrix and transformation of cercariae to schistosomula began within minutes of exposure to maximally stimulating concentrations of linoleic acid. Degradation continued for 24 hours and was dependent on the number of cercariae. Some degradation occurred without exogenous stimulants but at a slower rate than with skin lipid or linoleic acid. Degradation of matrix was inhibited by alpha 1-proteinase inhibitor and soybean trypsin inhibitor. Ethylenediaminetetraacetic acid inhibited degradation by unstimulated but not linoleic acid-stimulated cercariae. Preacetabular gland secretions collected from cercariae also degraded the matrix with an activity 86% of that of live cercariae. Preacetabular gland proteolytic activity was also inhibited by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, and ethylenediaminetetraacetic acid. The similar characteristics of matrix degradation by both live cercariae and cercarial preacetabular gland secretions support the idea that a proteinase secreted from cercarial preacetabular glands facilitates invasion of skin and connective tissue by these larvae. Degradation of elastin and glycoprotein constituents of extracellular matrix is probably essential for skin penetration.

Animals↗

PubMed1994

Heparin selectively inhibits synthesis of tissue type plasminogen activator and matrix deposition of plasminogen activator inhibitor 1 by human mesangial cells.

BACKGROUND: Mesangial changes in a variety of pathologic conditions involve mesangial cell proliferation and mesangial matrix remodelling. Heparin has been shown to prevent these processes in vivo. In vitro, heparin interferes with cell growth, proto-oncogene expression, synthesis of specific proteins, and extracellular matrix composition. In some cell types, it seems to interact with intracellular protein kinase C-dependent pathways. The effect of heparin on the mesangial plasminogen activating system (tissue type plasminogen activator, t-PA, and plasminogen activator inhibitor type 1, PAI-1), which is thought to be involved in matrix remodelling, has not been previously reported. EXPERIMENTAL DESIGN: Cultured human mesangial cells were stimulated by 10% fetal calf serum (FCS) or 16 nM phorbol myristate acetate (PMA) in the presence or absence of anticoagulant or nonanticoagulant heparins. Cell proliferation, synthesis of t-PA and PAI-1, cell morphology, and PAI-1 matrix deposition were studied using cell counting, [3H]thymidine incorporation, specific t-PA and PAI-1 enzyme-linked immunosorbent assay, Northern blot analysis, light microscopy, immunofluorescence and immunogold silver staining with combined bright-field and epipolarization microscopy. RESULTS: Heparin partially inhibited FCS-stimulated cell growth but not PMA-induced thymidine incorporation. FCS and PMA stimulated t-PA (p < 0.05 and p < 0.01, respectively) and PAI-1 synthesis (p < 0.05 and p < 0.01 respectively). Heparin selectively and partially inhibited FCS-stimulated t-PA, but not PAI-1 synthesis. It has no effect on PMA-stimulated t-PA or PAI-1 synthesis but prevented cell shape-changes induced by PMA, suggesting that heparin inhibits some but not all protein kinase C (PKC)-dependent effects and that heparin block in t-PA synthesis is distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar results were observed with anticoagulant and nonanticoagulant heparin fragments. CONCLUSIONS: In human mesangial cells, anticoagulant and nonanticoagulant heparin exert an antiproliferative effect and may prevent mesangial matrix changes by decreasing FCS-stimulated t-PA synthesis and PAI-1 deposition in the matrix. Heparin is able to inhibit PKC-dependent cell shape changes but not PKC-dependent t-PA or PAI-1 synthesis. It also inhibits PKC-independent cell proliferation and t-PA synthesis. These results suggest multiple intracellular sites of action for heparin, unrelated or distal to PKC activation.

Blotting, Northern↗

PubMed2004

Impact of collagen structure on matrix trafficking by human fibroblasts.

Biodegradation of collagen biomaterial matrices and the deposition of new collagen extracellular matrix (ECM) are critical to the integration of in vitro bioengineered materials and tissues in vivo. In previous studies, we observed significant impact of collagen matrix structure on primary lung fibroblast behavior in vitro. In the present work, to begin to understand the mechanistic basis for our previous observation, the response of human fibroblasts (IMR-90) to the structural state of collagen matrices was studied with respect to cell proliferation, cell morphology, beta-galactosidase level, and transcript content for collagen (Col-1), matrix metalloproteinases (MMP-1, MMP-2), tissue inhibitors of matrix metalloproteinase (TIMP-1 and TIMP-2). Collagen digestion was assessed quantitatively by uptake of collagen-coated fluorescent beads incorporated in the preformed collagen matrix. Transcript levels related to the deposition of new ECM proteins varied as a function of the structure of the collagen matrix presented to the cells. Col-1 expression was 2-fold higher and expression for MMP-1, MMP-2, TIMP-1, and TIMP-2 increased for cells when grown on 156 microg/cm2 denatured collagen compared with cells grown on tissue culture (TC) plastic. On 156 microg/cm2 nondenatured (native) collagen, Col-1 expression was decreased by half and MMP-2 was increased by 2.5-fold compared with cells grown on TC plastic. On 78 microg/cm2 denatured collagen, Col-1 expression was 80% whereas the MMPs and TIMPs were increased by 1.25- to 2-fold compared with cells grown on TC plastic. On 78 microg/cm2 nondenatured collagen expression of all 5 transcripts was reduced 60-90% of the levels determined for the cells grown on TC plastic. Cell viability, based on cell morphology and beta-galactosidase activity, was improved on the denatured collagen. A higher level of collagen matrix incorporation was observed for cells grown on denatured collagen than on nondenatured collagen or TC plastic. These data suggest that tissue engineering matrices incorporating denatured collagen may promote more active remodeling toward new ECM in comparison to cells grown on nondenatured collagen or cells grown on TC plastic.

Animals↗