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Biomedical subjects

D Gospodarowicz

Publications and source records attributed to D Gospodarowicz.

At least 127 records · Page 7Linked to original sources
PubMed1982

Fibroblast growth factor modulates synthesis of collagen in cultured vascular endothelial cells.

Vascular endothelial cells derived from adult bovine aortic arch can be grown in two ways, either in the presence or absence of fibroblast growth factor. The types of collagen produced by cultures under these two conditions have been compared. In the presence of fibroblast growth factor, cells grow in an orderly fashion, express their normal phenotype and synthesize primarily type III collagen plus collagens types IV and V at a ratio of 10:1:3. Cultures grown in the absence of the factor lose their orderly pattern of growth, lose polarity and normal phenotypic expression. They devote twice the proportion of total protein-synthesizing capacity to collagen, and now synthesize type I in addition to the other collagen types. The ratio of collagen types I:III:IV:V is approximately 30:70:1:13. The kinds of type V collagen chains expressed are also altered. Fibroblast growth factor appears to modulate collagen synthesis, the major component of the extracellular matrix, and indirectly modulates the phenotypic expression of cultured vascular endothelial cells. In atherosclerosis, type I collagen is found in association with the intimal layer. The disorderly growth and the abnormal production of type I collagen by these vascular endothelial cells cultured in the absence of fibroblast growth factor is a model for a number of pathological situations including atherosclerotic plaque formation.

Animals↗
PubMed1982

Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A reductase in progesterone production by cultured bovine granulosa cells.

The relative contributions of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to progesterone production by bovine granulosa cells exposed to plasma or liquor folliculi (LF) were studied. LF did not contain and very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), or low density lipoprotein (LDL). These lipoproteins were present in the plasma at concentrations of 92 micrograms protein/ml for VLDL and IDL together and 139 micrograms protein/ml for LDL. In contrast, high density lipoprotein (HDL) was present in LF at a concentration (763 micrograms protein/ml) that was 59% of that in plasma (1293 micrograms protein/ml). Bovine granulosa cells exposed to human plasma produce progesterone in response to dibutyryl cAmP. Sixty-three percent of the progesterone released by the cells was dependent on LDL but not HDL derived from human plasma. When cells were exposed to bovine plasma, 75% of the progesterone release was dependent on the presence of lipoproteins in the medium. Both LDL and HDL of bovine origin were able to support progesterone production, although LDL was effective at concentrations (on a molar basis) 20-fold lower than HDL. The LF was able to support progesterone production 45% as well as bovine plasma. The differences between the greater ability of the whole fractions and the lesser ability of their respective lipoprotein-deficient derivatives to support progesterone synthesis were 4-fold for bovine plasma, 2.7-fold for human plasma, and 1.7-fold for LF. The relative abilities of equivalent concentrations of LDL to restore the rate of progesterone synthesis seen in the lipoprotein-deficient fraction toward that seen in the whole fraction were greatest in the LF, intermediate in human plasma, and least in bovine plasma. These observations taken together suggest that the low level of support of progesterone synthesis that is offered by LF is due to its deficiency in LDL. HMG CoA reductase, the regulated and rate-limiting enzyme of cholesterol synthesis, was induced (2- to 3-fold) by dibutyryl cAMP and was suppressed by both human and bovine LDL and to a lesser extent by bovine HDL. Compactin, a competitive inhibitor of HMG CoA reductase, inhibited progesterone production relatively little when cells were exposed to complete plasma or LF. However, when cells were exposed to a lipoprotein-deficient bovine plasma or LF, compactin was very efficient in reducing (by 76%) progesterone release. Bovine granulosa cells exposed to plasma primarily use cholesterol derived from LDL in order to produce progesterone. Their ability to produce progesterone when exposed to LF was limited, and the cells were probably more dependent on de novo cholesterol synthesis than cells exposed to plasma.

Animals↗
PubMed1982

Proliferation and differentiation of bovine corneal endothelial cells in culture.

Bovine corneal endothelial cells can be grown successfully in culture. Growth factors such as FGF and EGF are shown to have a significant role in supporting an active proliferation of bovine corneal endothelial cells in culture as well as an effect on the ability of these cultures, to adopt a morphological configuration and phenotypic expression upon reaching confluence similar to their in vivo counterparts. Cultured bovine corneal endothelial cells provide us with an excellent in vitro model to study differentiated functions of the corneal endothelium. Using these cultures, we describe in the present review the exploration of a new differentiation property of the corneal endothelium such as the role of corneal endothelial cells in the inhibition and removal of active thrombin, as well as the study of a basic problem in cell biology such as the mechanism of action of growth factors.

Animals↗
PubMed1982

High-density lipoproteins and the proliferation of human tumor cells maintained on extracellular matrix-coated dishes and exposed to defined medium.

The ability of high-density lipoprotein (HDL) to support the growth of an established tumor cell line exposed to defined medium supplemented with transferrin has been examined. Low-density A-431 carcinoma cells maintained on extracellular matrix- or fibronectin-coated dishes proliferated actively when exposed to a synthetic medium supplemented with HDL, 500 micrograms protein per ml. Epidermal growth factor added at concentrations above 0.5 ng/ml inhibited cell growth, while at concentrations above 5 ng/ml it was cytotoxic. Among the various substrata tested for their ability to support the active proliferation of low-density A-431 cells when exposed to transferrin and HDL, plastic was the least efficient. On fibronectin-coated dishes, cells ceased to proliferate after 8 population doublings, while on extracellular matrix-coated dishes cells could be passaged for 50 population doublings. In the case of colon carcinoma, rhabdomyosarcoma, and Ewing's sarcoma cells exposed to medium supplemented with transferrin, the addition to the cultures of HDL alone resulted in a growth rate and final cell density which were similar to those observed when cells were exposed to serum-supplemented medium. In the case of the mammary carcinoma cell lines MCF-7 and ZR-75-1, HDL also supported cell growth, although to a lesser extent than did serum. The present study therefore indicates that HDL is capable of supporting, either totally or partially, the in vitro proliferation of tumor cells.

Blood↗
PubMed1982

Neurite outgrowth and protein synthesis by PC12 cells as a function of substratum and nerve growth factor.

Numerous studies have implied that enhanced cell-substratum adhesion plays a role in neurite outgrowth by neuronal cells. Using an extracellular matrix (ECM) produced by cultured corneal endothelial cells, we have investigated attachment and de novo neurite outgrowth by the pheochromocytoma cell line, PC12. PC12 cells were found to attach more rapidly and efficiently to the ECM than to plastic or collagen-coated surfaces. An extensive but temporary (5- to 10-day) neurite outgrowth occurred in the absence of nerve growth factor (NGF) when cells were on the ECM. However, long term neurite survival and further elongation required NGF. Our findings are consistent with the hypothesis that protein(s) in the ECM has an important role in neurite outgrowth. Thus, NGF may not so much initiate neurite outgrowth as it stabilizes neurites. ECM and NGF also were found to modulate cellular protein synthesis by PC12 cells. On ECM, a decreased synthesis of many high molecular weight proteins (Mr greater than 85,000) was observed in comparison to that of cells on collagen-coated dishes. The presence of neurites (in the presence of absence of NGF) as associated with the induction of the synthesis of a cellular protein (Mr = 55,000 to 56,000 and pI of 5.6). NGF was found to increase markedly the synthesis of two secreted proteins, while it drastically reduced the synthesis of all others regardless of the substratum upon which the cells were maintained.

Adrenal Gland Neoplasms↗
PubMed1981

Internalization and degradation of thrombin and up regulation of thrombin-binding sites in corneal endothelial cells.

Thrombin binds specifically to bovine corneal endothelial cells. Binding involves the formation of an apparently covalent complex between thrombin and its binding site, Mr = 77,000. The cells appear to internalize this complex by adsorptive endocytosis since there is a 10-fold greater amount of thrombin internalized than of prothrombin. Internalization proceeds at a rate of 4 ng of thrombin/1 X 10(6) cells/h and reaches a steady state by 2 h at 37 degrees C. Approximately 90% of the 125I-thrombin reappears in the extracellular media within 1 h of binding to the cells. Since this released 125I-labeled material cannot be precipitated by an anti-thrombin antibody or trichloroacetic acid, it probably represents degradation of thrombin into small peptides. Chloroquine treatment of the cells completely inhibits degradation of thrombin. This suggests that proteolysis occurs in lysosomes. Preincubation of corneal cells with physiological concentrations of thrombin for 2 to 24 h results in a concentration-dependent increase in synthesis and subsequent release into the incubation medium of thrombin binding sites. The increase in the rate of release of binding sites is proportional to the duration of pre-exposure of the cells to thrombin and reaches a maximal increase of approximately 6-fold at 24 h. 125I-thrombin binds to these soluble sites and forms a 77,000-dalton complex similar to that seen with the noninduced binding sites. This complex binds to the cells, is internalized, and then degraded. Binding, internalization, and degradation of thrombin by endothelial cells and the subsequent up regulation of binding sites may represent a mechanism for maintaining low extracellular levels of thrombin.

Animals↗
PubMed1981

Characterization of collagens synthesized by cultured bovine corneal endothelial cells.

Collagen is the major matrix protein produced by corneal endothelial cell cultures and represented 1% of total protein synthesis. The different types of collagen were determined by a combination of DEAE- and CM-cellulose column chromatography using known internal markers. Type III collagen was the major component both deposited in the extracellular matrix and secreted into the media. The basement membrane collagens, types IV and V, were also found in each compartment, although the latter was associated preferentially with the cell matrix. The ratios of types I:III:IV + V collagens synthesized by corneal endothelial cells were 3:16:1.

Animals↗
PubMed1981

Nuclear accumulation of epidermal growth factor in cultured bovine corneal endothelial and granulosa cells.

Intracellular accumulation of intact 125I-labeled epidermal growth factor (125I-EGF) in corneal or granulosa cell cultures exposed to either chloroquine or leupeptin can be 10-fold higher than that observed in cultures not exposed to the lysosomal inhibitors. This has made it possible to study the translocation and accumulation of 125I-EGF in a nuclear fraction of both cell types. The accumulation of 125I-EGF was found to be dependent on the inhibitor's concentrations. Chloroquine at a concentration of 5 X 10(-5) M yielded a maximal nuclear accumulation which amounted to 18% of the total 125I-EGF present within the bovine corneal endothelial cells exposed to chloroquine. Nuclear accumulation could be detected in that cell type as early as 4 h after cultures were exposed to both chloroquine and 125I-EGF and was maximal by 24 h. Saturation of nuclear accumulation of 125I-EGF in corneal endothelial and granulosa cell cultures was observed at 20 and 10 ng/ml of 125I-EGF, respectively. After 24 h, 1.06 X 10(4) and 1.4 X 10(4) 125I-EGF molecules were found associated with granulosa or corneal endothelial nuclei, respectively. In corneal endothelial cell cultures exposed to chloroquine, a small fraction (0.6%) of 125I-EGF associated with the cell was found to be irreversibly found to a polypeptide with a molecular weight of 185,000, and 50% of these irreversible 125I-EGF . receptor complexes were found to be associated with the nuclei.

Animals↗
PubMed1981

Respective roles of laminin and fibronectin in adhesion of human carcinoma and sarcoma cells.

In a previous study, Ewing's sarcoma cells and colon carcinoma cells failed to attach to plastic and to dishes coated with various collagen types I, III and IV, but adhered rapidly to extracellular matrix (ECM) produced by cultured corneal endothelial cells. However, although carcinoma cells required ECM and did not adhere to fibronectin, the sarcoma cells adhered and flattened almost equally well on either substrate. Thus, different adhesive proteins may mediate the attachment of sarcoma- and carcinoma-derived cells to extracellular matrices, the most likely being fibronectin and laminin. Fibronectin stimulates the adhesion of fibroblasts, but not epidermal cells, to collagen type IV (ref. 7) and could mediate the attachment of sarcoma cells. Laminin is confined to the lamina lucida region of basement membranes and has been localized to cellular adhesion sites. Studies of the attachment of epidermal cells in vitro (V.P. Terranova, personal communication) suggest that it is adhesive for epithelial cells, and so could have the same role for carcinoma cells as that played by fibronectin for sarcoma cells. Cultured vascular endothelial cells secrete fibronectin and laminin into both the ECM and the culture medium, and we report here that pre-exposure of plastic dishes to such conditioned medium induces the attachment and flattening of both human colon carcinoma and Ewing's sarcoma cells. While laminin mediates the attachment and spreading of the former fibronectin is responsible for the attachment and flattening of the latter.

Animals↗
PubMed1981

Covalent binding of thrombin to specific sites on corneal endothelial cells.

Binding of 125I-labeled human alpha-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 degrees C but to a much smaller extent at 4 degrees C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77000-dalton complex was demonstrated by NaDodSO4 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I]thrombin was blocked by a 100-fold excess of unlabeled alpha-thrombin and by the thrombin inhibitor, hirudin. There are approximately 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodSO4-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodSO4 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr approximately 90000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes (Mr less than 77000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that alpha-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-SO4-stable complex which requires the catalytically active enzyme.

Animals↗
PubMed1981

The production and localization of laminin in cultured vascular and corneal endothelial cells.

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical and basal cell surfaces, laminin does not ever seem to be present on the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.

Animals↗
PubMed1981

Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells.

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.

Animals↗
PubMed1981

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium.

Low density bovine vascular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL "in vivo."

Animals↗
PubMed1981

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture.

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.

Animals↗
PubMed1981

Effect of cell density on thrombin binding to a specific site on bovine vascular endothelial cells.

We studied thrombin binding to proliferating and confluent endothelial cells derived from bovine vascular endothelium. [125]thrombin was incubated with nonconfluent or confluent endothelial cells and both the total amount bound and the amount linked in a 77,000-dalton thrombin-cell complex were determined. Approximately 230,000 molecules of thrombin bound per cell in nonconfluent cultures compared to 12,800 molecules per cell in confluent cultures. Approximately 67,7000 thrombin molecules were bound in an apparently covalent complex, Mr = 77,000, with each cell in sparse cultures, whereas only 4,600 thrombin molecules per cell were bound in this complex with confluent cultures. Similar studies with [125I]thrombin and endothelial cells derived from bovine cornea revealed no difference either in the total amount of thrombin bound or in the amount bound in the 77,000-dalton complex using sparse or confluent cultures. When confluent vascular endothelial cultures were wounded, additional cellular binding sites for the 77,000-dalton complex with thrombin appeared within 24 h. A 237% increase in the amount of thrombin bound to these sites was induced by a wound which resulted in a 20% decrease in cell number in the monolayer. There was no significant increase in thrombin binding to other cellular sites at 24 h. These experiments provide evidence that the first change in thrombin binding after injury is an increase in the cellular sites involved in the 77,000-dalton complex, and suggest that thrombin binding to endothelial cells may be important in the vascular response to injury.

Animals↗