Search PubMed⌕ Search

Biomedical subjects

E A Jaffe

Publications and source records attributed to E A Jaffe.

At least 19 recordsLinked to original sources
PubMed1992

Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer.

Cell surface antigens of transformed cells are the traditional targets for antibody-guided detection and therapy of solid neoplasms. Alternative targets may be found in the tumor stroma, which contains newly formed blood vessels, reactive fibroblasts, and extracellular matrix proteins. The F19 cell surface glycoprotein of reactive fibroblasts is a prototypic stromal antigen since it is found in the stroma of > 90% of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors. In the present study, we define an additional tumor stromal antigen, FB5, that is selectively expressed in vascular endothelial cells of malignant tumors. Immunohistochemical analysis of 128 tumors identified FB5 in endothelial cells in 67% of the samples (including 41 of 61 sarcomas, 26 of 37 carcinomas, and 18 of 25 neuroectodermal tumors) whereas normal blood vessels and other adult tissues tested lacked FB5 expression. In vitro studies showed that FB5 is a M(r) 165,000 cell surface glycoprotein, comprised of a M(r) 95,000 core polypeptide and highly sialyated O-linked oligosaccharides but few if any N-linked sugars, and that the FB5 gene is located on chromosome 11q13-qter. The restricted tissue distribution of the FB5 protein, which we refer to as endosialin, suggests strategies for tumor imaging and therapy that are aimed primarily at the tumor vasculature.

Adult↗
PubMed1992

Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor.

Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the beta/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect beta/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the beta/A4 amyloid protein precursor via the secretory cleavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.

Acetylcholine↗
PubMed1992

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.

Bacterial Toxins↗
PubMed1992

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.

Amino Acid Sequence↗
PubMed1992

The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells.

Elevation of intracellular calcium in response to trypsin, bradykinin, thrombin or histamine is associated with a proportional increase in PGI2 production in cultured human umbilical vein endothelial cells (HUVEC), bovine pulmonary artery endothelial cells (CPAE), and bovine aortic endothelial cells (BAEC). The major agonists that induce increases in intracellular calcium and PGI2 production are thrombin and trypsin in HUVEC, bradykinin in CPAE, and bradykinin and trypsin in BAEC. These results suggest that endothelial cells derived from different species or sites require different agonists to induce increases in intracellular calcium and PGI2 production and that only agonists which increase intracellular calcium can stimulate PGI2 production.

Animals↗
PubMed1991

A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells.

Cleavage by thrombin of the platelet thrombin receptor exposes a new N-terminal segment SFLLRNPNDKYEPF (SFLL) which acts as a tethered ligand. The free peptide activates platelets and induces platelet aggregation. We now show that SFLL can also activate human umbilical vein endothelial cells (HUVEC) and induce rises in both cytosolic free calcium ([Ca2+]i) and prostacyclin (PGI2) production. These responses were time- and concentration-dependent and were similar to those for native thrombin except that they were not blocked by hirudin. Initial activation of HUVEC with thrombin desensitized the subsequent response to SFLL for both rises in [Ca2+]i and PGI2 production. Thus, SFLL alone can activate HUVEC and elevate [Ca2+]i and induce PGI2 production suggesting that the thrombin receptors on platelet and endothelial cells are functionally and structurally similar.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages.

We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO) after activation with interferon-gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occurring via pterin salvage. Moreover, SEP increased NO synthesis to greater than 150% of control values, suggesting that THB availability is rate-limiting for NO synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO synthesis by sepiapterin. Thus, cytokine-induced MBE NO synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO synthesis by cytokine-activated MBE was inhibited by NG-monosubstituted arginine analogs with a rank-order of potency NH2 greater than CH3 greater than NO2, in contrast with the rank-order of NO2 greater than NH2 greater than CH3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine-induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO synthase were observed with regard to calmodulin requirements. Whereas constitutive NO synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine-induced NO synthase was unaffected. In summary, NO synthesis by cytokine-activated MBE is THB-dependent, calmodulin-independent and inhibited by NG-substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages.

Amino Acid Oxidoreductases↗
PubMed1991

Phosphorylation of the proto-oncogene product eukaryotic initiation factor 4E is a common cellular response to tumor necrosis factor.

The initiation of mRNA translation is regulated by the reversible phosphorylation of several initiation factors. We report here that tumor necrosis factor-alpha (TNF) rapidly stimulates phosphorylation of one such factor, an mRNA cap binding protein, in several cell types which are important in vitro models of TNF action. This protein has been purified, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor 4E. These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the various actions of TNF in diverse cell types.

Amino Acid Sequence↗
PubMed1991

Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to cultured human endothelial cells.

Tumor necrosis factor (TNF), a potent inflammatory mediator secreted by monocytes during inflammation, was shown to significantly increase the adherence of Staphylococcus aureus to cultured human umbilical vein endothelial cells in vitro. The stimulatory effect of TNF was dose dependent and was bimodal with respect to time; bacterial adhesion peaked after 4 and 16 h of stimulation with recombinant human TNF-alpha. The ability of TNF-alpha to augment staphylococcal adherence to endothelial cells was contingent upon the presence of plasma factors. Thus, the complex interaction among cytokines (such as TNF), plasma factor(s), and the endothelium serves to modulate bacterial adherence to endothelial cells.

Bacterial Adhesion↗
PubMed1991

Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells.

The propensity of Staphylococcus aureus to cause acute endovascular infections during transient bacteremia is poorly understood. To examine the events leading to the attachment of staphylococci to endothelium, adherence assays were developed to study the role of blood factors in the mediation of staphylococcal adherence to cultured human umbilical vein endothelium in vitro. Results indicate that the preferential attachment of S. aureus to endothelial cells is mediated by fibrinogen adsorbed from plasma onto the endothelial surface. The binding is apparently specific because it could be blocked by goat anti-human fibrinogen antibody in a dose-dependent fashion while nonimmune goat IgG, mouse MAb against AG-1 (a platelet antigen found on the endothelial cell surface), nonspecific mouse MAb and rabbit antibodies to human vitronectin and fibronectin were not inhibitory. Our data also indicate that fibrinogen is a necessary but not the only blood constituent in the mediation of binding of S. aureus to endothelium. This was supported by the finding that fibrinogen alone, at a concentration equivalent to that in plasma, did not potentiate staphylococcal adherence as much as plasma while afibrinogenemic plasma reconstituted with fibrinogen did. Because fibrinogen is known to bind to endothelial cells, our data is consistent with the hypothesis that fibrinogen and additional plasma factor(s), possibly activated during inflammation, promote staphylococcal adherence to endothelium. In addition, the role of the fibrinogen binding receptor of S. aureus as an adherence factor to native endothelium is also suggested.

Antibodies↗
PubMed1990

Expression of functional thrombin receptors in xenopus oocytes injected with human endothelial cell mRNA.

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein endothelial cells. Oocytes injected with endothelial cell RNA responded to thrombin with a Ca2(+)-dependent depolarizing current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currents. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca2(+)-dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor.

Animals↗
PubMed1990

Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine.

Many cell types are known to synthesize nitric oxide (NO.) from L-arginine. There appear to be at least two forms of NO. synthase: an inducible, tetrahydrobiopterin- and flavin-dependent activity exemplified by the macrophage enzyme and a constitutive, Ca+(+)-dependent activity exemplified by the endothelial cell enzyme. L-NG-methylarginine inhibits NO. synthesis by both cell types. We now report that L-NG-aminoarginine and L-NG-nitroarginine are about 100-fold more potent than NG-methylarginine in blocking endothelial cell NO. synthesis. In contrast, NG-aminoarginine and NG-methylarginine are about equipotent with macrophages whereas NG-nitroarginine is much less potent. Since macrophage and endothelial cell NO. synthesis are differentially sensitive to the inhibitors, the panel of inhibitors can be used in complex biological systems to determine if macrophage-like or endothelial-like cells are the predominant source of NO.. Indeed, all three inhibitors elicit a strong pressor response in the anesthetized guinea pig, a result consistent with the view that endothelial cells continually produce vasodilatory NO(.).

Animals↗
PubMed1990

L-arginine, but not N alpha-benzoyl-L-arginine ethyl ester, is a precursor of endothelium-derived relaxing factor.

N alpha-benzoyl-L-arginine ethyl ester (BAEE) is a vasorelaxant which resembles an arginine-containing peptide; its action may be partially endothelium-dependent. Because L-arginine (ARG) has little potency as a vasorelaxant, it has been proposed that an arginine-containing peptide, rather than free ARG, is the immediate precursor of endothelium-derived relaxing factor/nitric oxide (EDRF/NO). In the present study we have characterized pharmacologically the vasorelaxant effect of BAEE and assessed the ability of BAEE to serve as a substrate for EDRF/NO synthesis. BAEE elicited a concentration-dependent vasorelaxation of guinea pig pulmonary artery (EC50 of 0.36 +/- 0.05 mM). This vasorelaxation was neither antagonized by -NG-methyl-L-arginine (100 microM), a competitive inhibitor of EDRF/NO synthesis from ARG, nor potentiated by superoxide dismutase (60 U/ml), a superoxide anion scavenger that prolongs EDRF lifetime. Additionally, compound LY 83583 (1 microM) and methylene blue (10 microM), inhibitors of soluble guanylyl cyclase, failed to block BAEE-induced vasorelaxation. Moreover, endothelium removal potentiated the vasorelaxant effect of BAEE 3-fold. Thus, BAEE-induced vasorelaxation is mediated by a direct action on vascular smooth muscle that is unrelated to EDRF/NO synthesis. Furthermore, ARG, but not BAEE, overcame the inhibition by NG-methyl-L-arginine of the acetylcholine-induced endothelium-dependent cyclic GMP accumulation in guinea pig aortic rings and of A23187-induced nitrite formation by cultured bovine aortic endothelial cells (nitrite is a convenient indicator of NO biosynthesis). Thus, BAEE cannot substitute for ARG as a substrate for EDRF/NO biosynthesis. Collectively, our findings support the notion that free ARG, rather than an arginine-containing peptide related to BAEE, is the immediate biosynthetic precursor of EDRF/NO.

Acetylcholine↗
PubMed1990

[Influence of lipopolysaccharide on angiotensin converting enzyme activity expressed by human umbilical vein endothelial cells in culture].

The activity of angiotensin converting enzyme (ACE) released into the culture medium of human umbilical cord vein endothelial cells in culture (HUVEC) for 24-hour incubation with or without various serotypes of lipopolysaccharide (LPS) was unchanged. ACE activity, however, in cell lysate or monolayer HUVEC after 24-hour incubation of various serotypes of LPS except LPS from E. coli, 026:B6, showed significant decrease, compared with no LPS treatment. Cell lysate contained much higher ACE activity than monolayer HUVEC after 24-hour incubation with or without LPS. These findings give rise to speculation that ACE or ACE-like substance might be located not only on the luminal surface, but also in cytoplasm of HUVEC, and also that decrease of ACE activity in the serum of septic adult respiratory distress syndrome (ARDS) patients might be due to reduced synthesis by vascular endothelial cells.

Animals↗
PubMed1989

IFN-gamma and IFN-alpha induce the expression and synthesis of Leu 13 antigen by cultured human endothelial cells.

Leu 13 is a 16 kDa human lymphocyte surface Ag and monoclonal anti-Leu 13 induces T cell aggregation and alters T cell proliferation. In previous studies using anti-Leu 13, Leu 13 Ag has been detected on endothelial cells (EC) of arteries, capillaries, and veins in human tissue sections. In contrast, in this study using the same antibody, Leu 13 Ag could not be detected on unstimulated cultured human umbilical vein endothelial cells (HUVEC) by indirect immunofluorescence, immunoperoxidase staining, or immunoisolation of radiolabeled Ag from labeled endothelial cells. However, when HUVEC were cultured with IFN-gamma, Leu 13 Ag was demonstrable using all three detection techniques. Leu 13 Ag was detectable after 24 h of incubation with IFN-gamma (30 U/ml) and was maximally expressed after 72 h. After removal of IFN-gamma, Leu 13 Ag progressively declined back to basal levels by 4 days. Induction of Leu 13 Ag expression by IFN-gamma was suppressed by cycloheximide. Although Leu 13 Ag expression was also induced by IFN-alpha, PMA, and IL-1 were inactive. When HUVEC were incubated with IFN-gamma, surface-labeled with 125I, and then solubilized, anti-Leu 13 immunoisolated a 16-kDa radioactive band that comigrated with Leu 13 immunoisolated from T cells. Similar results were obtained using HUVEC that were metabolically labeled with 35S-methionine. These results demonstrate that HUVEC stimulated with IFN-gamma and IFN-alpha express Leu 13 Ag on their surface and suggest that exposure of EC to IFN may induce EC to develop new functional properties at sites of inflammatory and immune reactions.

Antibodies, Monoclonal↗
PubMed1989

Selenium deficiency inhibits prostacyclin release and enhances production of platelet activating factor by human endothelial cells.

Selenium is an essential component of glutathione peroxidase, an enzyme which protects cells against peroxidation and controls concentrations of intracellular peroxides. Since selenium deficiency is clinically associated with an increased degree of atherosclerosis, the effects of selenium deficiency on prostacyclin (PGI2) and platelet activating factor (PAF) production by cultured human umbilical vein endothelial cells (HUVEC) were investigated. In selenium-deficient HUVEC, histamine-induced PGI2 synthesis was significantly decreased when compared to selenium-supplemented HUVEC; in contrast, histamine-induced PAF production was increased by selenium deficiency. Histamine-induced inositol trisphosphate and [Ca2+]i responses and the conversion of PGG2 and PGH2 to PGI2 were not altered by selenium deficiency. However, selenium deficiency decreased the conversion of exogenous arachidonate to PGI2 and markedly suppressed glutathione peroxidase activity. These results suggest that selenium deficiency, by decreasing glutathione peroxidase activity, makes HUVEC susceptible to peroxide-induced inhibition of the cyclooxygenase activity of PGH2 synthase, resulting in decreased PGI2 production. These changes may alter platelet function in vivo and thus play a role in the increased incidence of atherosclerosis reported in selenium-deficient individuals.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells.

The effects of thrombin and histamine on protein phosphorylation in intact cultured human umbilical vein endothelial cells (HUVEC) prelabeled with 32PO4 were investigated. Incubation of HUVEC with either thrombin or histamine, agonists known to induce rapid transient increases in intracellular calcium levels in HUVEC, caused a rapid reversible increase in the phosphorylation of a protein with a Mr = 100,000 independent of the presence of extracellular calcium. Immunological and biochemical studies demonstrated that this Mr = 100,000 protein is elongation factor 2 (EF-2), a substrate previously shown to be phosphorylated by calcium/calmodulin-dependent protein kinase III (Nairn, A. C., and Palfrey, H. C. (1987) J. Biol. Chem. 262, 17299-17303). EF-2 is crucial for protein synthesis because it catalyzes the translocation of peptidyl-tRNA on the ribosome. Phosphoamino acid analysis of the EF-2 immunoprecipitated from HUVEC revealed that all of the thrombin-stimulated phosphorylation occurred on threonine. EF-2 was also phosphorylated when HUVEC were treated with the calcium ionophore, ionomycin. Phosphorylation of EF-2 was not increased by treatment with D-Phe-Pro-Arg-chloromethyl ketone thrombin, phorbol dibutyrate, forskolin, or 8-bromo-cGMP. The transient nature of the phosphorylation of EF-2 is consistent with it having a role in mediating some of the transient effects of thrombin and histamine on endothelial cell protein synthesis and functional capabilities.

Calcium-Calmodulin-Dependent Protein Kinases↗