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Biomedical subjects

J R Gamble

Publications and source records attributed to J R Gamble.

At least 19 recordsLinked to original sources
PubMed1998

Early events in angiogenesis: cloning an alpha-prolyl 4-hydroxylase-like gene.

To gain further insight into angiogenesis we sought to clone genes which are actively expressed during this complex process. Using the Matrigel-induced in vitro model we were able to show that although several cell-types form reticular arrays of cells on the gels (align), only endothelial cells were able to go on and form the capillary-like structures reminiscent of patent vessels. Although this alignment process did not require gene activation we show that tube formation was ultimately dependent upon gene expression occuring during the first few hours that cells are seeded onto Matrigel. We generated a cDNA library enriched for the expression of those genes and have sequenced an alpha-prolyl 4-hydroxylase-like clone (angio 0.9). This clone shares 66% overall homology to the carboxy-terminal 106 amino-acids of the published human sequence. In the region corresponding to the co-factor binding domains, His 1 and His 2, angio 0.9 has >90% homology to the published sequence. Using an RNAse protection assay we show that the level of expression of the message of this clone is five fold elevated in endothelial cells which have aligned on Matrigel. The dependence of collagen, and collagen hydroxylation in angiogenesis is well documented. Thus, our results are demonstrable proof that the principle of this approach has the potential to generate novel discoveries.

Amino Acid Sequence↗
PubMed1997

Lumen formation during angiogenesis in vitro involves phagocytic activity, formation and secretion of vacuoles, cell death, and capillary tube remodelling by different populations of endothelial cells.

BACKGROUND: We have utilised an in vitro model of angiogenesis to investigate the morphological changes which occur during the formation of a lumen in capillary tubes. METHODS AND RESULTS: On collagen 1 gel in the presence of phorbol myristate acetate (PMA) and anti-alpha 2 beta 1 antibody, cell aggregation and alignment takes place within two hours of plating. The initial apparently homogeneous population of endothelial cells (EC) actually display at least three distinct phenotypes. One population, characterised by a phagocytic phenotype, migrated through the gel creating channels and defines the extent of the capillary network. These are later enveloped by a second population of cells characterised by intracellular vacuoles. The ultimate fate of these vacuoles is fusion with the plasma membrane. By 12 hours the original phagocytic cell population undergoes cell death, which morphologically appears apoptotic in nature. A consequence of the secretion of vacuoles and programmed cell death is the extensive remodelling of the capillary tubes, resulting in expansion of the intercellular space into a lumen. The remodelling results in 45% of the EC membrane contacting the lumenal surface at the expense of EC-EC and EC-matrix contact. A third population of cells implant between the EC involved in lumen formation and thus expand the size of the capillary tube. CONCLUSION: Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.

Antibodies↗
PubMed1997

Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood, synovial fluid, and synovial membrane in rheumatoid arthritis.

OBJECTIVE: To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments. METHODS: Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1beta (IL-1beta), IL-8, and tumor necrosis factor alpha (TNFalpha) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E2 (PGE2) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFalpha, IL-8, IL-1beta, and the IL-1 receptor antagonist protein (IL-1Ra). RESULTS: MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFalpha in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFalpha in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1beta and IL-1Ra or synovial fluid IL-1beta and PGE2 was found. CONCLUSION: MP therapy rapidly reduces IL-8 and TNFalpha levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFalpha expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.

Aged↗
PubMed1997

Regulation of leucocyte-endothelial interactions of special relevance to atherogenesis.

1. The cellular events underlying atherosclerosis include the accumulation of lipid-laden monocytes in the neointima. This process is associated with the expression of adhesion proteins and chemokines by the endothelium, in a manner similar to that seen after the administration of pro-inflammatory cytokines to endothelial cells. 2. The processes that limit endothelial responses to proinflammatory cytokines are, therefore, the subject of this paper. Evidence is presented that the cytokine TGF-beta exerts a tonic inhibitory influence on endothelial responses. Furthermore, the smooth muscle cells adjacent to endothelial cells have a similar effect to exogenous TGF-beta and this suggests that these two cells form a functional interactive unit. Finally, the atheroprotective lipid fraction, high-density lipoproteins (HDL), also inhibits endothelial activation. The mechanism of effect of HDL that appears separate from its traditional role in cholesterol transport may yield novel insights into atheroprotection.

Animals↗
PubMed1996

Transforming growth factor-beta 1 inhibits the production of IL-8 and the transmigration of neutrophils through activated endothelium.

A central mechanism of inflammation is the activation of vascular endothelium by the inflammatory cytokines TNF-alpha and IL-1. These cytokines induce the expression of adhesion molecules, the elaboration of chemokines, and the transendothelial migration of white cells. TGF-beta 1 has anti-inflammatory properties, is expressed in the vessel wall, and has previously been shown to inhibit leukocyte adhesiveness to the endothelium at least in part by inhibiting the expression of E-selectin. We now show that TGF-beta 1 also inhibits the migration of neutrophils through endothelial monolayers activated by TNF-alpha. At a dose of 10 U/ml TNF-alpha, the transmigration of neutrophils was inhibited 42.7 +/- 7.9% (n = 8) by 0.2 ng/ml TGF-beta 1. Furthermore, TGF-beta 1 inhibited, in a time- and dose-dependent fashion, the elaboration of IL-8 by TNF-activated endothelial cells by between 33 and 78% (TNF doses from 100 down to 0.1 U/ml) and the elaboration of mRNA for IL-8 by 69%. TGF-beta 1 treatment did not significantly alter the TNF-induced IL-8 mRNA stability, suggesting that the mechanism of action of TGF-beta 1 is on gene transcription. Neutrophil transmigration through cytokine-activated endothelium involves both IL-8-dependent and IL-8-independent mechanisms. Using an anti-IL-8 Ab, we show that TGF-beta 1 inhibits only the IL-8-dependent pathway, but does not affect the IL-8-independent transendothelial migration mechanism. These and our previous results show that TGF-beta1, achieves its anti-inflammatory properties by inhibiting the expression of at least two genes, E-selectin and IL-8, which are essential in the inflammatory pathway.

Cell Movement↗
PubMed1996

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3.

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

Cells, Cultured↗
PubMed1996

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.

Animals↗
PubMed1996

IL-3 receptor expression, regulation and function in cells of the vasculature.

IL-3 is a haemopoietic growth factor which stimulates the production and functional activity of various blood cell types. Recent evidence suggests that the target cell population of IL-3 is not restricted to haemopoietic cells as previously thought, but vascular cells such as endothelial cells also express receptors for and respond to this cytokine. Interestingly, IL-3 was found to regulate endothelial responses related to inflammation, immunity and haemopoiesis. These findings, summarized in this review, offer new insight into the physiological function of IL-3 and may also be of clinical importance, as IL-3 is used in bone marrow reconstitution following cancer therapy.

Cells, Cultured↗
PubMed1995

Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT.

Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.

Animals↗
PubMed1995

Interferon-gamma upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production.

The human interleukin-3 (IL-3) receptor is constitutively expressed on certain hematopoietic cells where it mediates proliferation and differentiation, or functional activation. We have recently found that human umbilical vein endothelial cells (HUVECs) also express IL-3 receptors and that the expression is enhanced by stimulation with the monokine tumor necrosis factor alpha. In this report we show that the lymphokine interferon gamma (IFN gamma) is a powerful stimulator of the IL-3 receptor of HUVECs and that the combination of IL-3 and IFN gamma has a synergistic effect on major histocompatibility complex (MHC) class II expression and on the production of the early-acting hematopoietic cytokines IL-6 and granulocyte colony-stimulating factor (G-CSF). IFN gamma caused a time- and dose-dependent up-regulation of mRNA for both the alpha and beta chains of the IL-3 receptor, with maximal effects occurring 12 to 24 hours after stimulation with IFN gamma at 100 U/mL. Induction of mRNA correlated with protein expression on the cell surface, as judged by monoclonal antibody staining of both receptor chains and by the ability of HUVEC to specifically bind 125I-labeled IL-3 (125I-IL-3). Scatchard analysis of HUVECs stimulated with IFN gamma at 100 U/mL for 24 hours showed approximately 6,300 IL-3 receptors per cell that were of a high affinity class (dissociation constant [kd] = 500 pmol/L) only. The addition of IL-3 to IFN gamma-treated HUVECs strongly enhanced the expression of MHC class II antigen. Importantly, IFN gamma and IL-3 also exhibited a synergistic effect in the induction of the mRNA for G-CSF and IL-6. This was reflected in increased amounts of G-CSF and IL-6 protein in HUVEC supernatants. In contrast, IFN gamma and IL-3 did not stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-8 production in HUVECs. These results show that IFN gamma is a strong stimulator of IL-3 receptor expression in HUVECs and suggest that in vivo T-cell activation, causing the concomitant production of IFN gamma and IL-3, may lead to enhanced endothelial MHC class II expression and to the selective production of early-acting hematopoietic cytokines. Thus, IL-3 could influence immunity and hematopoiesis by acting not only on hematopoietic cells, but also on vascular endothelium.

Drug Synergism↗
PubMed1995

Transendothelial migration of neutrophils involves integrin-associated protein (CD47).

Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.

Antibodies, Monoclonal↗
PubMed1995

Angiogenesis: models and modulators.

Angiogenesis in vivo is distinguished by four stages: subsequent to the transduction of signals to differentiate, stage 1 is defined as an altered proteolytic balance of the cell allowing it to digest through the surrounding matrix. These committed cells then proliferate (stage 2), and migrate (stage 3) to form aligned cords of cells. The final stage is the development of vessel patency (stage 4), generated by a coalescing of intracellular vacuoles. Subsequently, these structures anastamose and the initial flow of blood through the new vessel completes the process. We present and discuss how the available models most closely represent phases of in vivo angiogenesis. The enhancement of angiogenesis by hyaluronic acid fragments, transforming growth factor beta, tumor necrosis factor alpha, angiogenin, okadaic acid, fibroblast growth factor, interleukin 8, vascular endothelial growth factor, haptoglobin, and gangliosides, and the inhibition of the process by hyaluronic acid, estrogen metabolites, genestein, heparin, cyclosporin A, placental RNase inhibitor, steroids, collagen synthesis inhibitors, thrombospondin, fumagellin, and protamine are also discussed.

Animals↗
PubMed1995

High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules.

While an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-1 (VCAM-1) but also involves other molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 (IL-1) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-alpha-induced VCAM-1 and E-selectin are significantly reduced by physiological concentrations of HDLs. An an HDL concentration of 1 mg/mL apolipoprotein A-I, the protein expressions of VCAM-1, ICAM-1, and E-selectin were inhibited by 89.6 +/- 0.4% (mean +/-SD, n=4), 64.8 +/- 1.0%, and 79.2 +/- 0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-alpha receptor. HDLs were effective when added from 16 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-alpha-induced expression of ICAM-1 by human foreskin fibroblasts, suggesting that the effect is cell-type restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1995

TGF-beta and endothelial cells inhibit VCAM-1 expression on human vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are normally devoid of the adhesion protein vascular cell adhesion molecule-1 (VCAM-1), which has, however, been observed on human VSMCs in atheroma. We now show that cultured human saphenous vein VSMCs express small amounts of VCAM-1 and that the cytokine tumor necrosis factor-alpha (TNF-alpha) induces, in a time- and dose-dependent fashion, a significant increase in its expression. Interleukin (IL)-4, IL-1, and to a lesser extent interferon gamma have similar effects. TNF-alpha-stimulated human VSMCs demonstrate increased binding of T lymphocytes that is totally VCAM-1 mediated. The cytokine transforming growth factor-beta (TGF-beta) at 2.0 ng/mL inhibited basal VCAM-1 expression by 84 +/- 8% and the induction by TNF-alpha by between 56 +/- 16% and 77 +/- 15% depending on the dose of TNF. Furthermore, coculture on opposing sides of a polycarbonate filter of human VSMCs with human umbilical vein endothelial cells also inhibited the induction of VCAM-1 by 47 +/- 6%. As active TGF-beta is produced upon the coculture of VSMCs and endothelial cells, we suggest that the close physical proximity of these cells in vivo is responsible for the lack of expression of VCAM-1 on VSMCs and that the interruption of this contact in atheroma is an important pathogenic event. As VCAM-1 not only serves as an adhesion molecule but also as a costimulator of immune cells, its expression may be crucial in the propagation of vascular lesions.

Cell Adhesion↗
PubMed1994

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

The role of granulocyte-macrophage and granulocyte colony-stimulating factors in neutrophil transendothelial migration: comparison with interleukin-8.

Granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF) have proinflammatory effects on mature neutrophils. Both factors have been reported to cause neutrophil chemotaxis and are produced by cells stimulated by inflammatory mediators, including endothelial cells. We therefore tested the hypothesis that these factors might mediate neutrophil transendothelial migration, either by forming a gradient across the endothelial monolayer or through the production of CSFs by activated endothelium. Studies of neutrophil migration across filters without endothelium showed that migration was promoted in the presence of a gradient of either CSF, but was equally promoted against the gradient; that is, the CSFs are chemokinetic but not chemotactic. The CSFs promoted migration of neutrophils across endothelial monolayers cultured on filters, but the magnitude of this effect was very small compared with that of a prototypic neutrophil chemoattractant, interleukin-8 (IL-8) (migration index [stimulated/unstimulated] 1.8-fold for GM-CSF, 10.8-fold for IL-8). Activation of endothelial monolayers by preincubation with tumor necrosis factor-alpha (TNF-alpha) increased neutrophil transmigration significantly; neutralizing antibodies to IL-8 inhibited this increase by 44%, whereas neutralizing anti-GM-CSF antibodies did not inhibit it. These data suggest little role for the CSFs in neutrophil diapedesis at inflammatory sites in vivo. Exposure of neutrophils to GM-CSF decreased their migration through TNF-activated monolayers, whereas G-CSF did not. This may have implications for the therapeutic administration of these factors.

Antibodies↗
PubMed1994

Characterization of a spontaneously transformed human endothelial cell line.

BACKGROUND: A line of cells was isolated from a focus observed in a human umbilical vein endothelial cell (HUVEC) culture, presumably the result of a spontaneous transformation event. EXPERIMENTAL DESIGN: The cell line was continuously cultured for 16 months, after which time, the proliferative rate, capacity to be cloned, and ability to be transfected was investigated. The cell line was analyzed for expression of endothelial cell markers, von Willebrand Factor, P selectin, and scavenger receptor. We also examined the tumor necrosis factor (TNF)-mediated upregulation of E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. We evaluated the levels of expression and types of TNF-alpha receptor expressed by the cell line, and the cell lines response to interleukin-4 and interferon-gamma. CD34 expression of this cell line and the ability of transforming growth factor-beta to inhibit the TNF-alpha induction of E-selectin was examined. The ability to support neutrophil adhesion and transmigration, and generate capillary-like tubes in vitro was assessed. Finally, the karyotype and tumourigenicity of this line was established. RESULTS: This cell line (C11STH) has a doubling time comparable to that of normal HUVECs and has a trisomy of chromosome 8 and 11. The cell line is capable of generating colonies at clonal density, and is transfectable with efficiencies comparable to normal HUVECs. C11STH expresses von Willebrand factor, P-selectin, and scavenger receptor to an extent similar to passaged HUVECs and can be induced to express E-selectin, VCAM, and ICAM with TNF-alpha. C11STH expresses both the p55 and p75 subunits of TNF-alpha receptor at levels similar to HUVECs. The ability of interleukin-4 to enhance the expression of VCAM and reduce the TNF-alpha-mediated expression of E-selectin is maintained in this cell line. C11STH cells are unable to induce class II major histocompatibility antigen in response to interferon-gamma. However, interferon-gamma is able to synergize with TNF to enhance the expression of E-selectin. C11STH cells do not express CD34 or show transforming growth factor-beta inhibition of TNF-alpha induced E-selectin expression, functions indicative of primary, or early passage HUVECs. The cell line retains the ability to support neutrophil adhesion and transmigration and can generate patent tubes when seeded onto complex basement membrane gels. However, the cell line no longer has the ability to generate capillary-like vessels when seeded onto collagen gels in the presence of phorbol 12-myristate 13-acetate. CONCLUSIONS: C11STH has retained many of the normal characteristics of endothelial cells, is able to proliferate at clonal density, and is easily transfectable. The line will provide a useful resource for endothelial cell biology. Its ability to make capillary-like tubes on basement membrane gels, but not on collagen will provide a powerful tool with which to further analyze the processes involved in angiogenesis, and will enable us to define the role of specific proteins in angiogenesis. Since C11STH shows tube competence on Matrigel, but is not tube competent on collagen, our studies suggest that capillary-tube formation on Matrigel and collagen occur via qualitatively different mechanisms. Thus, this cell line provides the opportunity to examine the signalling mechanisms required to generate capillary tube formation. These may include the involvement of matrix molecules, the production of proteases and inhibitors, gene regulation and kinases or phosphatases.

Cell Adhesion↗