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Biomedical subjects

D Gospodarowicz

Publications and source records attributed to D Gospodarowicz.

At least 163 records · Page 9Linked to original sources
PubMed1980

Extracellular matrix and control of proliferation of vascular endothelial cells.

Bovine vascular endothelial cells plated at low cell density in the presence of high (10%) concentrations of serum and maintained on plastic tissue culture dishes proliferate slowly. If the cultures were exposed to fibroblast growth factors (FGF), the cells proliferated actively and, after a week, a monolayer composed of closely apposed and highly contact-inhibited mononucleated cells formed. In contrast to cultures maintained on plastic, cultures maintained on dishes coated with an extracellular matrix produced by corneal endothelial cells proliferated rapidly and no longer required FGF to reach confluence. Addition of FGF to such cultures did not decrease the mean doubling time, which was already at a minimum (18 h), nor did it result in a higher final cell density, which was already at a maximum (700-1,000 cells/mm(2)). Likewise, although human umbilical vein endothelial cells plated at low density on plastic did not proliferate, they proliferated rapidly when plated on dishes coated with an extracellular matrix. However, unlike bovine vascular endothelial cells, they still required FGF if the cultures were to become confluent. The ability of plasma vs. serum to sustain cell proliferation was analyzed using low density bovine-vascular endothelial cell cultures maintained either on plastic or on dishes coated with an extracellular matrix. Cells plated on plastic had a lower growth rate when exposed to plasma than to serum. In both cases, FGF was required for the cultures to become confluent. In contrast, when cells were plated on an extracellular matrix, they proliferated equally well, regardless of whether they were exposed to plasma or serum, and no longer required FGF to become confluent. Because the growth rate of the cultures maintained on an extracellular matrix was a direct function of the serum or plasma concentrations to which they were exposed, it is likely that the extracellular matrix had a permissive rather than a direct mitogenic effect on the cells. Therefore, one can conclude that the simple change of substrate from plastic to extracellular matrix will restore the sensitivity of vascular endothelial cells to physiological agents present in plasma or serum.

Animals↗
PubMed1980

Effect of high and low density lipoproteins on proliferation of cultured bovine vascular endothelial cells.

Bovine vascular endothelial cells maintained on dishes coated with an extracellular matrix and exposedto medium supplemented with lipoprotein-deficient serum (LPDS) require the presence of lipoprotein to proliferate optimally. High density lipoprotein (HDL) seems to be the major factor involved in the proliferation of vascular endothelial cells. This is mostly due to its lack of toxicity when added at high concentration, as well as to its nondependence on LPDS to exhibit its mitogenic properties. Therefore, HDL at physiological concentrations (1,000--1,500 microgram protein/ml) can fully replace serum. Low density lipoprotein, unlike HDL, has a biphasic effect. Although mitogenic for vascular endothelial cells when added at low concentration, once physiological concentrations are reached it becomes toxic for the cells. Moreover, and in contrast with HDL, the mitogenic effect of low density lipoprotein was found to be a function of the LPDS concentration to which cultures were exposed. The substrate upon which cultures are maintained has been found to be an important factor if a mitogenic effect of HDL is to be observed. When maintained on plastic, cells proliferate poorly in response to HDL unless fibroblast growth factor is added to the medium. In contrast, when maintained on extracellular matrix, an optimal growth rate is induced by HDL, even in the absence of fibroblast growth factor. This suggests that, in vivo, the integrity of the basement membrane upon which endothelial cells rest and migrate is an important factor in determining the cells response to lipoproteins present in plasma.

Animals↗
PubMed1980

Patterns of cellular peptide synthesis by cultured bovine granulosa cells.

The electrophoretic distribution of the polypeptides synthesized by bovine granulosa cell cultures after metabolic labeling with [35S]methionine has been analyzed by double gel electrophoresis. The fluctuations of 35 polypeptides have been followed as a function of the size of the follicles from which cultures originated, as a function of the cultures' proliferative stage (sparce, actively growing vs. confluent, resting cultures), and finally as a function of whether cells were exposed to either epidermal or fibroblast growth factor. When the patterns of protein synthesis in sparce vs. confluent granulosa cell cultures derived from small-sized follicles were compared, only a few differences were observed. In confluent cultures, 6 new peptides appeared, while 1 peptide present in sparce cultures disappeared. Cultures maintained in the presence of fibroblast or epidermal growth factor synthesized 20 new peptides upon reaching confluence. Among these were the 6 new peptides present in confluent but not in sparse granulosa cell cultures maintained in the absence of growth factors. The changes in protein synthesis observed in cultures grown in the presence of growth factors may reflect their direct effect on the cellular metabolism. A comparison between the protein distribution in cells derived from small- vs. large-sized follicles showed that fewer proteins were ultimately produced at confluence in cells derived from large-size follicles than in cells derived from small-sized follicles. This could be related to the process of cellular differentiation taking place within granulosa cells. The patterns of [35S]methionine-labeled proteins secreted by the granulosa cells into the incubation medium were analyzed and found to be similar regardless of the size of the follicle from which the culture originated. Little similarity between the proteins present in the follicular fluid and the pattern of the labeled proteins secreted into the incubation medium by cultured granulosa cells was observed. Only three proteins were identified which comigrate with proteins present in the follicular fluid. One of these was identified as fibronectin. This raises the possibility that the fibronectin present in the follicular fluid originated from granulosa cells and is not derived from plasma.

Animals↗
PubMed1979

Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.

Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.

Animals↗
PubMed1979

Effect of contact inhibition on the regulation of cholesterol metabolism in cultured vascular endothelial cells.

Cholesterol synthesis in actively growing bovine vascular endothelial cells is regulated by low density lipoprotein (LDL) at a step prior to mevalonate formation, in a manner comparable to that found in aortic smooth muscle cells. LDL uptake by these cells is associated with induction of cholesterol esterification, an increase in total cell cholesterol, and an inhibition of endogenous sterol synthesis. In contrast, cholesterol metabolism in confluent contact-inhibited endothelial cultures was not significantly affected by LDL even though the cells bind the lipoprotein at high affinity receptor sites. Lysosomal degradation and subsequent regulatory effects on cellular cholesterol metabolism, however, were observed in contact-inhibited endothelial cells incubated with cationized rather than native LDL. Cationized LDL enter the cells independently of the high affinity sites. Therefore, the primary regulation of cholesterol metabolism in these cells is neither through the appropriate intracellular enzymes nor through the high affinity surface receptors, but via an inhibition of LDL internalization. It is suggested that this inhibition is due to a strict contact-inhibited morphology which enables the endothelium of the larger arteries to function as a selective barrier to the high circulating levels of plasma LDL.

Animals↗
PubMed1979

Transplantation of cultured bovine corneal endothelial cells to species with nonregenerative endothelium. The cat as an experimental model.

The in vivo transplantation of cultured bovine corneal endothelial cells has been attempted in the cat. Cat corneas denuded of their endothelium were coated with bovine corneal endothelial cells previously maintained in tissue culture. When grafted back into cat recipients, the corneal buttons remained clear with no edema. Alizarin red staining of the endothelial side of the corneal transplant demonstrated that the coated bovine corneal endothelial cells reorganized themselves into a highly organized cell monolayer within eight days in vivo. In contrast, corneas denuded of their endothelium became opaque and edematous within seven days and remained so thereafter. These results demonstrate that cultured corneal endothelial cells remain functional in vitro and can replace a damaged or nonfunctional endothelium i

Animals↗
PubMed1979

Serum-dependent regulation of alpha-aminoisobutyric acid uptake in bovine granulosa cells.

The removal of serum from the medium of ovarian granulosa cells in exponential or confluent stages of growth results in a rapid and pronounced decrease in the rate of transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid. This decrease is rapidly and completely reversed by the addition of serum. The decrease and its reversal are insensitive to inhibitors of RNA and protein syntheisis and are unaffected by a number of other metabolic inhibitors. The serum requirement cannot be replaced by peptide hormones known to stimulate cell division and secretion by these cells. These data are consistent with a model of post-translational control of AIB transport by a high-molecular-weight component of serum.

Aminoisobutyric Acids↗
PubMed1979

Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution.

Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistrubution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cells independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization. The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.

Cell Division↗
PubMed1979

Structural and functional alterations in the surface of vascular endothelial cells associated with the formation of a confluent cell monolayer and with the withdrawal of fibroblast growth factor.

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) divide actively when seeded at low or clonal cell densities and upon reaching confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vivo. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentiation. At confluence the endothelial cells form a monolayer of closely apposed and nondividing cells that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low-density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP-60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic appearance and physiologic behavior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP-60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cells. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentiation and phenotypic expression of the vascular endothelium.

Animals↗