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Biomedical subjects

N Savion

Publications and source records attributed to N Savion.

At least 109 records · Page 6Linked to original sources
PubMed1985

Binding and processing of trypsin by cultured bovine corneal endothelial cells.

Bovine corneal endothelial cells in culture bind internalize and degrade [125I]-trypsin. Binding involves the active site of trypsin and increases as a function of [125I]-trypsin concentration. Saturation is observed at a concentration of 0.5-1.0 micrograms ml-1. The cell surface binding of [125I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [125I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [125I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [125I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [125I]-trypsin X protease-nexin with a molecular weight of 64000 daltons. The cells, when incubated at 37 degrees C, appear to internalize the cell surface-bound [125I]-trypsin at a rate of 0.15-0.25 ng (10(6) cells)-1 min-1. Both the non-covalently cell surface-bound and the protease-nexin (PN) mediated-bound [125I]-trypsin are internalized by the cells, but the [125I]-trypsin X PN complexes contribute about 75% of the total amount of [125I]-trypsin internalized by the cells. The internalized [125I]-trypsin is degraded by the cultures at a rate of about 0.05 ng (10(6) cells)-1 min-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [125I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [125I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.

Amyloid beta-Protein Precursor↗
PubMed1984

Interaction of T lymphocytes and macrophages with cultured vascular endothelial cells: attachment, invasion, and subsequent degradation of the subendothelial extracellular matrix.

Circulating macrophages and T lymphocytes can invade the vascular endothelium and migrate from the circulatory system to an extravascular compartment such as inflammatory organs. In an in vitro model system we have examined the capacity of murine T lymphocytes and peritoneal macrophages to attach and invade a confluent vascular endothelial cell monolayer and to degrade sulfated proteoglycans in the subendothelial extracellular matrix. Concanavalin A and antigen-specific (egg albumin) activated T lymphocytes labeled with [3H]thymidine attached to the apical surface of the vascular endothelium in a time-dependent manner. A subsequent invasion of the endothelial cell monolayer was observed by scanning electron microscopy. Both activated T lymphocytes and murine macrophages degraded the [35S]O4 = -containing fragments in a process which required cell-matrix contact but was not dependent on serum proteases. Sulfated glycosaminoglycan chains produced from matrix proteoglycans by treatment with papain or alkaline borohydride were 3-4 times larger than the cell-mediated degradation fragments. This suggests that both macrophages and T lymphocytes elaborate upon stimulation an endoglicosidase capable of cleaving glycosaminoglycans specifically and releasing heparan sulfate-rich fragments. The ability of activated cells of the immune system to attach and invade the vascular endothelium and to degrade sulfated proteoglycans is very similar to that reported for highly metastatic tumor cells.

Animals↗
PubMed1984

Binding uptake and degradation of antithrombin III X protease complexes by cultured corneal endothelial cells.

Interaction of 125I-labeled human antithrombin III (125I-AT III) X protease complexes with bovine corneal endothelial cells has been studied in tissue culture. 125I-AT III does not bind to endothelial cells, but its complexes with either thrombin or trypsin bind specifically to the cultures. The binding of 125I-AT III X protease complexes is not via the moiety of the free antithrombin III (AT III) or the free protease, since neither AT III nor thrombin compete on the binding of 125I-AT III X thrombin complexes. Only unlabeled AT III X thrombin complexes compete on the binding of the iodinated ligand. 125I-AT III X trypsin complexes bind with a KD of 1.4 X 10(-7) M to high affinity-binding sites present on the cell surface of corneal endothelial cells. Saturation of binding to the cell surface is observed at a concentration of 2.5 X 10(-7) M 125I-AT III X trypsin complexes and the number of binding sites per cell is about 4 X 10(4). The cell surface binding reaches a maximum by 15 min and then decreases with time. The cells, when incubated at 37 degrees C, appear to internalize the bound complexes by adsorptive endocytosis which proceeds at a rate of 0.5-0.8 pmole/1 X 10(6) cells/h. The internalization process of 125I-AT III X protease complexes is saturated at a concentration of 2.5 X 10(-7) M. Since the cells release 125I-labeled material into the extracellular media which cannot be precipitated by trichloroacetic acid (TCA), it probably represents degradation of 125I-AT III X protease complexes into small fragments at a linear rate of about 0.5 pmole/1 X 10(6) cells/h. The described process of AT III X protease complexes binding, internalization and subsequent degradation by corneal endothelial cells may represent a clearing mechanism for extracellular AT III X protease complexes formed under pathological conditions.

Animals↗
PubMed1983

Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli.

Cultured bovine corneal endothelial cells can be grown in three ways: on plastic, on plastic with fibroblast growth factor present in the media, and on their own preformed extracellular matrix. On plastic alone, cells grow in a disorderly fashion and secrete matrix on all cell surfaces. Cells grown on plastic with growth factor or on a matrix, at confluence, have matrix deposition only on the basal surface of the cells and an orderly contact-inhibited pattern of growth. This correlates with the polarity they demonstrate histologically. This cell-matrix pattern resembles the pattern observed in vivo. Both the soluble growth factor and the extracellular matrix are able to modulate the pattern of collagen synthesis and deposition by cells, but they do so in two entirely different ways. In cells grown on the extracellular matrix, total collagen synthesis is lower but more efficient. Collagen is deposited primarily into the cell layer even at the early sparse stage of culture. In cells grown on plastic with growth factor in the media, collagen is initially secreted into the media and does not become incorporated into the matrix. The deposition of collagen on the basal surface of cell occurs only late in the culture, and is achieved by increments in a stepwise manner. The in vivo-like pattern is not manifest until confluence has been reached. Thus, the extracellular matrix functions not only as a structural support, but is also instructional to the cells plated on it. In this case, the matrix regulates the level of collagen synthesis in the cells and modulates the pattern of collagen deposition. Soluble growth factors may act in part by enhancing a cell's ability to elaborate an appropriate matrix pattern necessary for the cell's own growth and accurate function.

Animals↗
PubMed1983

Cultivation, proliferation and characterization of thymic macrophages.

Successful long-term culture of murine thymic macrophages was achieved by plating adherent thymic cells, in the presence of L cell-conditioned medium, on dishes coated with an extracellular matrix. Adherent thymic cells in normal conditions of in-vitro culture do not proliferate. Those maintained on plastic tissue-culture dishes, and exposed to L cell-conditioned medium, proliferate slowly to a limited degree and form very small colonies. In contrast, when cultured in dishes coated with an extracellular matrix formed by corneal endothelial cells, in the presence of L cell-conditioned medium, adherent thymic cells proliferate rapidly and after 12-21 days in culture form large colonies (about 3-5 mm in diameter). The proliferating cells were identified to be mononuclear phagocytes by their morphological appearance, their ability to ingest both bacteria and antibody-coated erythrocytes and by their nonspecific esterase activity. These cells were also shown to exhibit cell surface antigens that are characteristic of differentiated macrophages, e.g. Fc receptors and the specific macrophage cell surface marker F4/80. A high percentage of these cultured cells were found to bear I-A antigens. The adherent thymic mononuclear phagocytes could be trypsinized and passaged while maintaining both their ability to proliferate and their specific macrophage characteristics for a period of 70 days. Thus, monocyte-macrophage stem cells ae present in the thymus, and under appropriate in-vitro conditions, can be made to proliferate and mature to I-A-bearing macrophages.

Animals↗
PubMed1982

Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and organization.

Confluent cultures of adult bovine aortic endothelial (ABAE), corneal endothelial (BCE), and fetal bovine heart endothelial (FBHE) cells form a monolayer of highly flattened, closely apposed, and nonoverlapping cells. In ABAE and BCE cultures, this is associated with a 50-fold decrease in the rate of DNA sythesis and correlates with a 14-fold decrease in protein synthesis. In contrast, in confluent FBHE cultures only partial decreases in the rates of DNA synthesis (6-fold) and protein synthesis (3-fold) are observed. FBHE cells therefore fulfill the morphological, but not the biochemical, criteria for confluent cultured endothelial cell monolayers. The appearance of the cytoskeletal elements actin, tubulin, and vimentin in sparse and confluent cultures of endothelial cells has been analyzed by two-dimensional gel electrophoresis and immunofluorescence. Sparse versus confluent ABAE, FBHE, and BCE cultures showed no changes in their relative rates of synthesis or cellular content of tubulin. Actin behaved similarly to tubulin in FBHE and BCE cultures, while in ABAE cultures a small increase (3-fold) in its relative rate of synthesis was observed in confluent versus sparse cultures. BCE cultures showed no change in the rate of synthesis of vimentin, but the cellular content of vimentin was markedly increased when cultures reached confluence. When the distribution of vimentin in both sparse and confluent BCE cultures was analyzed by immunofluorescence, in both cases it appeared distributed throughout the cytoplasm as thin fibers and bundles of fibers. In confluent ABAE cultures, both the relative amount and biosynthetic rate of vimentin increased by 15-fold. This increase in the intracellular accumulation of vimentin correlated with its immunofluorescent distribution within the cells. While in sparse cultures, vimentin appeared to be distributed as thin fibers, in confluent cultures thick curl-like fibrous bundles could be seen distributed throughout the cytoplasm and organized in a perinuclear ring. In contrast, in FBHE cultures no significant changes in the distribution and organization or rate of synthesis of vimentin were observed.

Actins↗
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

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

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↗