7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, alpha-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

Abstract

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, α-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

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.     

Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells

Chlamydophila pneumoniae is a common respiratory pathogen that has been shown to be associated with coronary artery disease. Recent studies have shown that one of the possible mechanisms of the atherogenicity of C. pneumoniae is overexpression of cell adhesion molecules (CAMs) in infected endothelial cells. We investigated whether exposure of C. pneumoniae-infected endothelial cells to oxidized LDL (oxLDL) leads to further upregulation of CAMs. Flow cytometry and immunoblot analysis of human aortic endothelial cells (HAECs) was performed for intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ICAM-1 was expressed in 78.7% of C. pneumoniae-infected HAECs. The addition of oxLDL (100 microg/ml) to infected HAECs increased the proportion of ICAM-1-positive cells to 92%. VCAM-1 was only observed in 9.3% of infected HAECs, and the addition of oxLDL had no further effect on the surface expression of VCAM-1. C. pneumoniae also upregulated the surface expression of E-selectin on 52.2% of the cells, and incubation with oxLDL further increased the proportion of positive cells to 63.64%. In conclusion, C. pneumoniae upregulated the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin on HAECs. The addition of oxLDL to the infected cells further enhanced the surface expression of ICAM-1 and E-selectin.     

The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-alpha

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.     

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE^-/-) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.     

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.     

Oxygen tension regulates neutrophil adhesion to human endothelial cells via an LFA-1-dependent mechanism

Extravasation of leukocytes at the sites of ischemia-reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO₂ of 50 torr for 3 hours, increased 2.5-fold, 2-, and 1.5-fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell-associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia-induced adhesion was inhibited by LFA-1-specific (CD 11 a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM-1 ligand for the LFA-1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA-1 molecule on edothelial cells which can be affected by hypoxic environments.     

Insulin administration in the mild hyperglycaemia changes expression of proinflammatory adhesion molecules on human aortic endothelial cells

An overexpression of cell adhesion molecules (CAMs) on the surface of endothelial cells is one of the first steps in a high glucose-mediated endothelial dysfunction in diabetic patients. The effect of insulin administration in the condition of elevated glucose concentration on the E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) expression on human aortic endothelial cells (HAEC) was investigated. Cells were cultured for 4 h in a medium supplemented with homocysteine (7 pM) and different concentration of glucose (5.5, 8.0, 12.0 and 16.5 mM respectively) with or without insulin (1 mlU/mL) addition. Expression of CAMs was analysed by flow-cytometry using monoclonal antibodies. Controls were CAMs expression in the medium with a corresponding glucose concentration. Obtained results show that short-term exposure of HAECs to moderate high glucose concentrations results in increased expression of E-selectin (2-fold), VCAM-1 (3-fold) and ICAM-1 (47%). At the same time, HAEC grown with 12 mM glucose expressed lesser E-selectin and, more ICAM-1 (for 64%) and VCAM-1 (41%) molecules. 16.5 mM glucose decreased expression of all investigated adhesion molecules. Addition of insulin was not changed expression of CAMs in a medium with 5.5 mM glucose. In conditions of elevated glucose concentration (12 mM), addition of insulin significantly dropped E-selectin (27%) and increased VCAM-1 (23%) expression. In conclusion, moderate elevated glucose concentration increased expression of cell adhesion molecules on HAEC. Insulin administration in the mild hyperglycaemia reduces an expression of the proinflammatory adhesion molecule E-selectin which could contribute in deceleration of macrovascular complications development in diabetic patients.     

Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage

The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin α_IIbβ₃-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of β₃ placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.