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Biomedical subjects

E A Jaffe

Publications and source records attributed to E A Jaffe.

At least 55 records · Page 3Linked to original sources
PubMed1986

Binding of plasminogen to cultured human endothelial cells.

Endothelial cells are known to release the two major forms of plasminogen activator, tissue plasminogen activator (TPA) and urokinase. We have previously demonstrated that plasminogen (PLG) immobilized on various surfaces forms a substrate for efficient conversion to plasmin by TPA (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human PLG to cultured human umbilical vein endothelial cell (HUVEC) monolayers, utilizing a newly devised cell monolayer enzyme-linked immunosorbent assay system. PLG binding to HUVEC was concentration dependent and saturable at physiologic PLG concentration (2 microM). Binding of PLG was 70-80% inhibited by 10 mM epsilon-aminocaproic acid, suggesting that it is largely mediated by the lysine-binding sites of PLG. PLG bound at an intermediate level to human fibroblasts, poorly to human smooth muscle cells, and not at all to bovine smooth muscle or bovine endothelial cells; unrelated proteins such as human albumin and IgG failed to bind HUVEC. PLG binding to HUVEC was rapid, reaching a steady state within 20 min, and quickly reversible. 125I-PLG bound to HUVEC with an estimated Kd of 310 +/- 235 nM (S.E.); each cell contained 1,400,000 +/- 1,000,000 (S.E.) binding sites. Functional studies demonstrated that HUVEC-bound PLG is activatable by TPA according to Michaelis-Menten kinetics (Km, 5.9 nM). Importantly, surface-bound PLG was activated with a 12.7-fold greater catalytic efficiency than fluid phase PLG. These results indicate that PLG binds to HUVEC in a specific and functional manner. Binding of PLG to endothelial cells may play a pivotal role in modulating thrombotic events at the vessel surface.

Animals↗
PubMed1986

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells.

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.

Cells, Cultured↗
PubMed1986

Oxygen-independent inhibition of intracellular Chlamydia psittaci growth by human monocytes and interferon-gamma-activated macrophages.

We have demonstrated previously that Chlamydia psittaci grows well in human monocyte-derived macrophages, but to a limited extent in lymphokine-or interferon-gamma (IFN-gamma)-activated macrophages. In this investigation, freshly explanted human monocytes inhibited chlamydial inclusion formation by 85% as compared to macrophages, and the level of inhibition was similar to that exhibited by lymphokine-activated macrophages (79%). To determine whether the oxygen-dependent antimicrobial mechanisms of the mononuclear phagocyte were involved in the inhibition, cells were infected with C. psittaci in the presence of agents that either inhibit the respiratory burst (glucose deprivation) or diminish the effect of H2O2 (catalase). These treatments had no effect on the capacity of monocytes and lymphokine-activated macrophages to restrict chlamydial growth. In addition, monocytes and activated macrophages from an individual with chronic granulomatous disease suppressed chlamydial growth as effectively as normal cells. Oxidatively deficient HeLa and endothelial cells, once stimulated by lymphokine, also displayed normal levels of antichlamydial activity. The induction of this apparently oxygen-independent antichlamydial effect by lymphokine was completely neutralized by a monoclonal anti-IFN-gamma antibody, and could be achieved by treatment with recombinant (r)IFN-gamma alone. These results indicate that the primary antimicrobial mechanism of the human monocyte against C. psittaci is oxygen-independent, and that this response can be effectively stimulated in the macrophage by lymphokine (IFN-gamma).

Antibodies, Monoclonal↗
PubMed1986

Decay-accelerating factor is present on cultured human umbilical vein endothelial cells.

Decay-accelerating factor (DAF) has been previously described only in cells of bone marrow origin where it serves as a negative modulator of complement activation. Using mAb against human DAF, we demonstrated the presence of DAF in human umbilical vein endothelial cells by immunofluorescence microscopy and flow cytometry. By means of an immunoradiometric assay we detected an average of 3.3 X 10(5) molecules of DAF on each cell. When immunoisolates were analyzed in Western blots, endothelial cell DAF comigrated with DAF purified from normal erythrocytes. DAF was synthesized by the endothelial cells since 35S-labeled DAF could be immunoisolated from HUVEC cultured in medium containing [35S]methionine. This is the first evidence for the presence of DAF in cells of extra-marrow origin. DAF may protect endothelial cells from complement-mediated injury.

Blood Proteins↗
PubMed1985

Biosynthesis of functionally active heparin cofactor II by a human hepatoma-derived cell line.

Human plasma heparin cofactor II (HCII) inhibits thrombin by rapidly forming a stable, equimolar complex in the presence of heparin or dermatan sulfate. Cultured human hepatoma-derived cells (PLC/PRF-5) secreted (approximately equal to 200 ng/ml in 3 days) a protein of MW - 72 kD that was immunoisolated and immunoblotted with anti-HCII, co-migrated on SDS-PAGE with human plasma HCII, and formed covalent complexes with thrombin (MW - 101 kD) in the presence but not absence of heparin or dermatan sulfate; these complexes co-migrated with those obtained by incubating thrombin with human plasma under the same conditions. HCII was not detectable (less than 0.13 ng/ml) in post-culture medium from cultured human umbilical vein endothelial cells or human foreskin fibroblasts.

Antibody Specificity↗
PubMed1985

Self-regulation of procoagulant events on the endothelial cell surface.

Interleukin 1 (IL-1) is a potent mediator of inflammatory and immunologic phenomena. In addition, IL-1 may be intimately involved in the regulation of hemostasis, since interaction of IL-1 with endothelial cells has been reported to induce tissue factor activity. We demonstrate that perturbation of the endothelial cell induces augmented IL-1 release. Human umbilical vein endothelial cells perturbed by treatment with lipopolysaccharide produced enhanced amounts of IL-1 activity. IL-1 activity from lipopolysaccharide-treated endothelial cell supernatants could be absorbed by an antibody to IL-1 coupled to Sepharose. Elaboration of IL-1 activity was dependent on the dose of lipopolysaccharide and occurred in a time-dependent manner. Addition of cycloheximide blocked generation of IL-1 activity. A physiological vessel wall perturbant, the coagulation enzyme thrombin, induced comparable amounts of IL-1 activity in endothelial cell cultures. This effect was specific for the enzyme, since active site-blocked thrombin and prothrombin had no effect on IL-1. In addition, IL-1-containing supernatants from thrombin-stimulated endothelial cells induced tissue factor procoagulant activity in fresh endothelial cell cultures. Thus, in contrast to the multiple, known inhibitory mechanisms that block thrombin procoagulant activity, these data suggest a circle of interaction in which thrombin induces endothelial cell elaboration of IL-1, a mediator of endothelial cell procoagulant activity. Endothelial cell production of IL-1 in response to perturbation allows these cells to play an integral role in the regulation of the inflammatory and coagulation systems.

Animals↗
PubMed1985

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations.

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.

Adipose Tissue↗
PubMed1985

Prostacyclin production by cultured endothelial cell monolayers exposed to step increases in shear stress.

While fluid shear stress is an important cardiovascular factor in vivo, it has generally been ignored in in vitro assays of endothelial cell function. We quantified the influence of shear stress on the production of prostacyclin by confluent monolayers of bovine aortic endothelial cells placed in a lucite flow chamber and exposed to flowing culture medium at constant shear stress at 37 degrees C and pH 7.4. Continuous inverted-phase microscopy (x 300) of the monolayers showed no significant contraction or detachment of cells under these conditions. Step increases in shear stress from zero to 14 dyne/cm2 caused rapid rises in prostacyclin production, from a baseline (n = 4) of 0.17 +/- 0.062 ng/cm2.min (mean +/- SEM) to peak values within 2 minutes, followed by a decline over several minutes. Peak prostacyclin production increased (p less than 0.005) with shear stress, from 0.60 +/- 0.13 ng/cm2.min at 0.9 dyne/cm2 (n = 14) to 2.33 +/- 0.67 ng/cm2.min at 14 dyne/cm2 (n = 10). The time integral of production or total production, however, did not significantly change with shear stress at least for shear stresses above 0.9 dyne/cm2. Once stressed, cell monolayers produced additional prostacyclin in response to stimulation by Na arachidonate or the calcium ionophore A23187, but not to repeat mechanical stimulation. We conclude that endothelial cells produce bursts of prostacyclin in response to suddenly imposed arterial-like shear stress, and that the peak rate, but not the time integral, of this production increases with shear stress.

Animals↗
PubMed1985

Monocytes and macrophages synthesize and secrete thrombospondin.

Thrombospondin, one of the major glycoproteins released from alpha-granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate-polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.

Animals↗
PubMed1984

Endothelial cells contain beta adrenoceptors.

The direct identification of beta adrenoceptors in endothelial cell cultures has not been possible until the advent of a new beta-adrenergic radioligand, [125I]iodocyanopindolol ([125I]ICYP). Using [125I]ICYP, we report the successful identification of a beta adrenoceptor in cultured bovine aortic endothelial cells. At 37 degrees C, specific binding is saturable, stable and reversible. There is a single class of binding sites (21,500 +/- 2,900 sites/cell) with an equilibrium dissociation constant (Kd) of 109 +/- 26 pM. The rate constant of association, k1, is 1.22 X 10(9) M-1 min-1 and of dissociation, k-1, is 0.01 min-1. Binding studies on monolayers of endothelial cells grown in microtiter plates yield similar data (Kd = 53 +/- 9 pM, Bmax = 20,000 +/- 1,900 sites/cell). Stereoselectivity of binding for the (-)-isomer is demonstrable for both agonists and antagonists. A series of adrenergic agonists competes with [125I]ICYP for binding with an order of potency suggesting beta 2 subselectivity ; isoproterenol (0.73 microM) greater than epinephrine (15 microM) greater than norepinephrine (71 microM). Furthermore, the beta 2 inhibitor butoxamine is more potent than the beta 1 inhibitor practolol (7.7 microM vs 22 microM respectively). The GTP analogue, Gpp(NH)p, reduces isoproterenol affinity to 1.9 microM and increases the Hill coefficient from 0.62-0.90.

Adrenergic beta-Agonists↗
PubMed1984

A membrane antigen (HC1) selectively present on hairy cell leukemia cells, endothelial cells, and epidermal basal cells.

A monoclonal antibody (alpha HC1) raised against HCL cells reacted specifically with HCL cells and not with other normal or leukemic B cell types. It also reacted with BC and with vascular EC. One case of Kaposi's sarcoma, a tumor thought to derive from EC, was also stained by this antibody. Sinusoidal cells of liver and spleen and EC in the brain did not stain with alpha HC1, but EC from all other tissues and EC in culture were positive. Because the exact normal cell equivalent of the leukemic hairy cell is unknown, similarities in phenotype and function between EC and hairy cells are of particular interest.

Antibodies, Monoclonal↗
PubMed1983

Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.

Thrombospondin, a major glycoprotein released from alpha granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 micrograms per 10(6) cells per 24 hr, respectively); secretion was blocked by cycloheximide. [3H]Thrombospondin was isolated from [3H]leucine-labeled fibroblast postculture medium and from cell layers with rabbit polyclonal or mouse monoclonal anti-thrombospondin coupled to staphylococcal protein A-Sepharose. The immunologically isolated [3H]thrombospondin migrated in NaDodSO4/polyacrylamide gels with purified marker platelet thrombospondin both with and without reduction. Immunofluorescence microscopy using rabbit polyclonal and mouse monoclonal anti-thrombospondin antibodies localized thrombospondin to the fibrillar extracellular matrix surrounding the cells. Thus, cultured human fibroblasts secrete thrombospondin and incorporate it into the extracellular matrix.

Cells, Cultured↗
PubMed1982

Synthesis and secretion of thrombospondin by cultured human endothelial cells.

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.

Cells, Cultured↗
PubMed1982

Plasminogen activator and collagenase production by cultured capillary endothelial cells.

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4alpha-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.

Animals↗