The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.     

Alterations in the Expression of ELAM-1, ICAM-1 and VCAM-1 after In Vitro Infection of Endothelial Cells with a Clinical Isolate of Human Cytomegalovirus

Human cytomegalovirus (HCMV) infection of endothelial cells resulted in increased adhesion of the cells to peripheral blood leukocytes. It was demonstrated by flow cytometry that increased adhesiveness parallels the increased expression of cell surface adhesion molecules (ELAM-1, ICAM-1, VCAM-1). The increased adhesion of PMN and T-lymphocytes was due to upregulation in the expression of ELAM-1 and ICAM-1. The upregulation of VCAM-1 resulted in the increased adhesiveness of monocytes and T-lymphocytes to HCMV-infected HUVEC. The increased adhesiveness to leukocytes was caused by HCMV replication since endothelial cells exposed to HCMV-free supernatants and UV-inactivated HCMV did not show any increase in adhesiveness to any of the leukocytes tested.     

Stimulation of mononuclear cell binding to human endothelial cell monolayers by thrombin

The common occurrence of fibrin deposits in chronic inflammatory lesions suggests a possible role for thrombin in the mobilization of mononuclear cell infiltrates. For this reason, the effect of thrombin on the binding of mononuclear cells to endothelial cells (EC) was investigated. Incubation of confluent monolayers of human umbilical vein endothelial cells with thrombin markedly enhanced EC adhesiveness for both T lymphocytes and U937 cells (a monocyte-like cell line) in a time- and dose-dependent fashion. This effect was EC specific: 1) treatment of the T cells or the U937 cells with thrombin did not stimulate their adherence to EC, and 2) treatment of human foreskin fibroblasts with thrombin did not stimulate their inherently low adhesiveness for T cells. Fixation of EC monolayers with paraformaldehyde after pre-incubation with thrombin did not affect the increased adhesiveness for T cells. mAb against the LFA-1 antigen (mAb 60.3 (anti-CD18) or mAb TS1/22 (anti-CD11a), which inhibit the binding of T cells to unstimulated EC, failed to block the increased adhesion induced by thrombin, indicating that the increased binding induced by thrombin is similar to that induced by IL-1 and TNF, which showed similar resistance. These results suggest that thrombin may have a role in the extravascular emigration of mononuclear cells from post-capillary venules by virtue of its ability to stimulate the adhesiveness of EC for both lymphocytes and monocytes.     

Ionizing radiation increases adhesiveness of human aortic endothelial cells via a chemokine-dependent mechanism

Exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Since radiation also induces inflammation, a possible mechanism is a change in the adhesiveness of vascular endothelial cells, triggering pro-atherogenic accumulation of leukocytes. To investigate this mechanism at the cellular level, the effect of X rays on adhesiveness of cultured human aortic endothelial cells (HAECs) was determined. HAECs were grown as monolayers and exposed to 0 to 30 Gy X rays, followed by measurement of adhesiveness under physiological shear stress using a flow chamber adhesion assay. Twenty-four hours after irradiation, HAEC adhesiveness was increased, with a peak effect at 15 Gy. Radiation had no significant effect on surface expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Antibody blockade of the leukocyte integrin receptors for ICAM-1 and VCAM-1, however, abolished the radiation-induced adhesiveness. Since these leukocyte integrins can be activated by chemokines presented on the endothelial cell surface, the effect of pertussis toxin (PTX), an inhibitor of chemokine-mediated integrin activation, was tested. PTX specifically inhibited radiation-induced adhesiveness, with no significant effect on nonirradiated cells. Therefore, radiation induces increased adhesiveness of aortic endothelial cells through chemokine-dependent signaling from endothelial cells to leukocytes, even in the absence of increased expression of the adhesion molecules involved.     

Disparate effects of tumor necrosis factor-alpha/cachectin and tumor necrosis factor-beta/lymphotoxin on hematopoietic growth factor production and neutrophil adhesion molecule expression by cultured human endothelial cells

Tumor necrosis factor-alpha/cachectin (TNF-alpha) and tumor necrosis factor-beta/lymphotoxin (TNF-beta) are inflammatory mediators with similar spectrums of cytotoxic activity against tumors in vitro and in vivo. We compared the effect of purified recombinant human TNF-alpha and TNF-beta on neutrophil adhesion molecule expression and hematopoietic growth factor production by cultured human umbilical vein endothelial cells. Endothelial cells acquired adhesive properties for neutrophils after a 4-hr incubation with as little as 5 U/ml TNF-alpha. TNF-alpha stimulated a dose-dependent increase in endothelial cell adhesiveness for neutrophils, with a maximal effect at 250 U/ml. In contrast, TNF-beta did not enhance endothelial-dependent neutrophil adherence until a concentration of 600 to 1200 U/ml was reached. Endothelial cells cultured for 24 hr with TNF-alpha, 10 to 1,000 U/ml, released hematopoietic colony-stimulating activity. TNF-beta failed to augment growth factor production by endothelial cells at any concentration tested. Inhibitor assays showed that the absence of detectable colony-stimulating activity was not due to direct inhibition of colony growth by TNF-beta or to release of hematopoietic inhibitors by the TNF-beta-stimulated endothelial cells. Purified natural TNF-beta was similar to recombinant TNF-beta in its effect on neutrophil adhesion molecule expression and growth factor production by endothelial cells. These results indicate that the two immunomodulatory proteins TNF-alpha and TNF-beta differ in their effects on a common target tissue. TNF-beta, which retains tumoricidal properties, shows fewer proinflammatory activities on cultured endothelial cells than TNF-alpha in vitro.     

Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1.

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes.

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.     

Accessory cell function of human endothelial cells. I. A subpopulation of Ia positive cells is required for antigen presentation

The expression of class I and class II HLA antigens on preparations of human endothelial cells, isolated from umbilical cord veins, was investigated by immunofluorescence. While virtually all endothelial cells expressed class I antigens, less than 1% were positive for class II antigens, as detected with a panel of 10 different monoclonal antibodies. Antigen specific T cell lines proliferated in response to mumps antigen in the presence of endothelial cells or blood monocytes from HLA-DR matched donors. However, these T cell lines failed to respond in the absence of accessory cells or when accessory cells from HLA-D-region mismatched cord donors were used. The ability of both monocytes and endothelial cells to present antigen was abolished by treatment of the cells with monoclonal antibodies specific for either class I or class II HLA antigens plus complement. Similar treatment with monoclonal antibodies specific for monocytes greatly reduced antigen presentation by endothelial cells. These results indicate that preparations of endothelial cells contain a subpopulation of Ia positive cells, distinct from monocytes, which are required for antigen presentation.     

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.     

Lovastatin induces differentiation of Mono Mac 6 cells

The proliferation of human monocytic Mono Mac 6 cells was significantly retarded by treatment with lovastatin (LOV, 10 μM) for 72 h. Treatment of Mono Mac 6 cells with LOV increased surface protein expression of monocyte-associated CD14 and the integrin-chain CD11b towards levels found in isolated human blood monocytes. These effects were dose-dependent and completely reversed by the isoprenoid precursor mevalonate (MVA). LOV failed to induce growth retardation and upregulation of CD11b or CD14 in the less mature premonocytic U937 cell line. While CD11b expression was comparable in Mono Mac 6 cells treated with LOV (10 μM), TNF (100 U ml^?1) or LPS (10 ng ml^?1), upregulation of CD14 by LOV was less pronounced. Basal CD23 expression was unaffected by LOV but markedly reduced by treatment with TNF or LPS. Moreover, LOV enhanced Mono Mac 6 adhesiveness to human umbilical vein endothelial cells to levels found in isolated human blood monocytes, probably due to the increased CD11b and CD14 expression. In conclusion, LOV can induce differentiation of monocytic cells which is reflected by the retardation of growth, expression of CD14 and CD11b, and enhanced adhesiveness.     

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.     

Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.     

Increased adhesion to fibronectin and Mo-1 expression by diabetic monocytes

As excessive monocyte adhesion to blood vessel wall could produce endothelial cell injury, we undertook comparative studies on the monocyte adhesiveness in insulin-dependent diabetics with vascular complications (n = 26) and healthy, normal subjects (n = 36). In the diabetic group, the extent of monocyte adhesion on fibronectin- and autologous plasma-coated surfaces was significantly increased compared with that in the control group (p less than 0.01 on both types of surfaces). Monocytes adhesion to the plasma-coated surface, but not to the fibronectin-coated surface, could be inhibited by monoclonal anti-Mo-1 antibody in a dose-dependent manner. For diabetic monocyte adhesion, a higher amount of anti-Mo-1 antibody was required to produce a similar extent of inhibition as observed with control monocytes. This indication of increased Mo-1 expression on diabetic monocytes was further confirmed by analyzing the fluorescence intensity of monocytes labeled with the antibody anti-Mo-1. The results of the present study suggest that diabetic monocytes have increased adhesiveness as the result of increased expression of fibronectin and Mo-1 receptors.     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

Interleukin 1 stimulates endothelial cells to release multilineage human colony-stimulating activity

Cultured human umbilical vein endothelial cells, when exposed to soluble products of peripheral blood monocytes, elaborate granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). We have performed studies to determine if the monokine IL 1 can stimulate endothelial cells to release hematopoietic growth factors and whether such factors can also support human megakaryocyte (Meg) and mixed-cell colony growth. Various concentrations of recombinant human IL 1 beta (rIL 1) and media conditioned by monocytes (MCM), endothelial cells (ECM), and endothelial cells cultured for 3 days in 50% MCM (ECMM) or rIL 1 (ECMrIL 1) were added to marrow mononuclear cells cultured in methylcellulose. ECMM and ECMrIL 1 stimulated, in a dose-dependent fashion, the growth of Meg, mixed-cell, and GM colonies and erythroid bursts. In contrast, ECM, MCM, and rIL 1 displayed little or no activity in the colony-forming assays. Preincubation with specific antisera to native human IL 1 or rIL 1 reduced by 75 to 100% the activity of MCM in stimulating endothelial cell release of BPA, GM-CSA, Meg-CSA, and mixed-cell CSA. Meg-CSA, although readily detectable at ECMM and ECMrIL 1 concentrations in culture of 1 to 5%, was partially masked by lineage-specific inhibitors of Meg colony growth. When ECMM was analyzed by gel filtration chromatography, the megakaryocytopoietic inhibitory activity eluted in the high Mr fractions (greater than 75 kD). Meg-CSA co-eluted with GM-CSA and BPA in a single peak of 30 kD. We conclude that endothelial cells, in response to IL 1, produce one or more growth factors that probably act on multiple classes of progenitor cells.     

Anti-proliferative effects of calcitriol on endothelial cells derived from two different microenvironments

Calcitriol (1,25-dihydroxycholecalciferol), the active form of Vitamin D, is anti-proliferative in tumor cells and tumor-derived endothelial cells (TDEC). However, endothelial cells isolated from normal tissues as cell lines or freshly isolated cells or from implanted Matrigel plugs (MDEC) are relatively resistant. Both TDEC and MDEC express similar amounts of Vitamin D receptor (VDR) protein. Although the VDR from TDEC has higher binding affinity for calcitriol than those from MDEC, VDR in both cell types translocates to the nucleus and transactivates the 24-hydroxylase promoter-luciferase construct. Calcitriol selectively inhibits the growth of TDEC but not MDEC by inducing G(0)/G(1) cell cycle arrest and by promoting apoptosis. This selectivity appears to be related to 24-hydroxylase (CYP24) expression. Calcitriol significantly induced CYP24 expression in MDEC but not in TDEC and inhibition of CYP24 activity in MDEC restores their sensitivity to calcitriol. These findings indicate that the induction of CYP24 expression differs in endothelial cells isolated from different microenvironments (TDEC versus MDEC) and that this distinction contributes to selective calcitriol-mediated growth inhibition in these cell types.