Endothelial Cell Extracellular Matrix Research Articles

Binding of heparin to human endothelial cell monolayer and extracellular matrix in culture.

Endothelial binding of heparin contributes to its local antithrombotic action and catabolism. However, it is uncertain whether heparin may be bound to a damaged or even de-endothelialized vessel surface. Therefore, the binding of 3H-heparin to cultured endothelial cell monolayer and extracellular matrix was studied. The binding reached equilibrium after 4 h. 3H-heparin bound to endothelium could be displaced by unlabelled heparin which competed for about 80% of binding. The binding became saturable when the concentration of heparin exceeded 30-times the therapeutical value. Approximately 6 X 10(11) binding sites for heparin per cm2 of endothelium were calculated. Heparin was bound not only to endothelial cells but also to extracellular matrix, even when it was exposed in the absence of cells. About 40% of binding sites were localized in the extracellular matrix fraction. A similar affinity of binding of 3H-heparin to complete endothelium, endothelial cells and extracellular matrix was estimated /Kd of almost 1 microM/. The binding of heparin to extracellular matrix should be considered in the interpretation of heparin interaction with endothelium.

Extracellular matrix derived from astrocytes stimulates neuritic outgrowth from PC12 cells in vitro

The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.

Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.

Macrophages stimulate DNA synthesis in rat alveolar type II cells.

Proliferation of alveolar type II cells after lung injury is crucial for repair of the epithelium. Because an influx of macrophages occurs as part of the inflammatory response associated with acute lung injury and macrophages produce mitogenic factors for a variety of cell types, experiments were conducted to determine if macrophages stimulated DNA synthesis in type II cells. Dialyzed medium conditioned by macrophages consistently stimulated type II cell DNA synthesis, whereas medium conditioned by a variety of other cell types did not. A SV40-transformed macrophage cell line, produced in our laboratory, also secreted substance(s) that enhanced 3H-thymidine incorporation into type II cells. In addition, coculturing rat alveolar macrophages with type II cells stimulated DNA synthesis in the epithelial cells. As determined by autoradiography, the addition of macrophages to type II cells cultured on plastic or on an endothelial cell extracellular matrix increased the labeling index of the epithelial cells from 1 to 15% and from 19 to 51%, respectively. The culture conditions that promoted the greatest increase in DNA synthesis, as well as an increase in cell number, occurred with type II cells plated on an extracellular matrix in medium containing macrophage-conditioned medium, cholera toxin, insulin, and epidermal growth factor. The results suggest that substances secreted by macrophages play a role in regulating alveolar type II cell proliferation in vivo.

Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80–90 kD as determined by affinity-labelling with [ 3 H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5–8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125 I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.

The effect of the extracellular matrix on the detachment of human endothelial cells

Human umbilical vein endothelial cells can be serially passaged by supplementing medium with a partially purified growth factor. Cell-substratum detachment of early and late passage endothelial cells was examined using trypsin, collagenase, or homocysteine. Late-passage cells detached more rapidly than early passage cells under all conditions tested. The rate of detachment was dependent upon the specific agent used. Protease-mediated detachment was most rapid, occurring over minutes, in contrast to homocysteine-induced detachment, which occurred over hours. When detached cells were collected and replated in the absence of the detaching agent, these cells reattached, spread, and continued to proliferate. No significant difference was observed in the rate of adhesion of either early or late passage cells to a gelatin matrix. When early or late-passage endothelial cells were plated and grown to confluence on a matrix synthesized by the opposite cell type, the rate of protease-mediated cell detachment resembled the cell type from which the matrix was derived. The ease of endothelial cell detachment was determined by the origin of the extracellular matrix. Examination of the extracellular matrices from early and late passage cells revealed significant differences in the amounts of glycosaminoglycans and sulfated proteins present. These studies demonstrate the importance of the endothelial cell extracellular matrix in protease-mediated cell detachment. The rate of cell detachment was controlled by the extracellular matrices are not altered by the endothelial cells.

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro.

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.

Von Willebrand protein binds to extracellular matrices independently of collagen.

Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the presence of alpha, alpha'-dipyridyl, a collagen-secretion inhibitor. These results indicate that the presence of von Willebrand protein in the extracellular matrix of endothelial cells and the binding of von Willebrand protein to foreign matrices do not depend on the matrix collagen.

How culture conditions modulate the morphofunctional differentiation of the human estradiol-sensitive mammary cell line (MCF-7)

The MCF-7 cell line grown on plastic surfaces is widely accepted as a model for hormone sensitivity in molecular biology. However, in vitro results concerning estrogen sensitivity remain controversial. In search of culture conditions most closely simulating the in vivo microenvironment we cultured MCF-7 cells on diverse substrates and in suspension culture. The different factors of the contact environment: (A) influence of diffusive medium, (B) influence of cell to cell contacts, and (C) influence of cell to substrate contacts were considered. Using morphological criteria:phase contrast microscopy, scanning and transmission electron microscopy we observed MCF-7 morphofunctional differentiation under the different culture conditions. Plastic, corneal endothelial cell extracellular matrix, and attached collagen gels imposed a planar medium-aggregate interface. The impermeability of the free surface and the intense basal tension antagonized epithelial polarization. Only at post-confluence did domes and clusters appear above the monolayer. On floating collagen gels and in suspension culture the cells established intimate cell-cell contacts over large surfaces and reconstituted tissular architecture. Three-dimensional growth conditions which approach the in vivo contact environment of epithelial cells should be used instead of the traditional monolayer cultures for assessing hormonal and pharmacological responses of human breast carcinomas.