Abstract
The endothelial expression of adhesion molecules by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been suggested to contribute to the initiation of atherosclerotic plaque formation. Since lactosylceramide (LacCer) accumulates in large quantities in human atherosclerotic plaque, we have explored its role in TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs). We found that TNF-alpha increased LacCer synthesis by way of stimulating the activity of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase in a time-dependent fashion. The TNF-alpha-induced expression of ICAM-1 was abrogated by D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase. However, the addition of LacCer reversed the D-PDMP effect on TNF-alpha-induced ICAM-1 expression in human umbilical vein endothelial cells. Northern hybridization analysis of mRNA levels and enzyme-linked immunosorbent assays revealed that LacCer (5 microM) specifically stimulated ICAM-1 at both the transcriptional and translational levels. This was accompanied by the adhesion of PMNs, which was visualized by confocal microscopy. Further studies revealed that LacCer stimulated the endogenous generation of superoxide radicals (O-2) about 5-fold compared with the control by specifically activating plasma membrane-associated NADPH-dependent oxidase. This phenomenon was blocked by the antioxidant N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, and the NADPH oxidase inhibitor, diphenylene iodonium. Overexpression of endogeneous CuZn-superoxide dismutase via an adenoviral vector carrying cDNA for CuZn-superoxide dismutase, also inhibited LacCer-induced ICAM-1 expression in endothelial cells. In sum, our findings suggest that LacCer may play the role of a lipid second messenger in TNF-alpha-induced pathogenesis by activating an oxidant-sensitive transcriptional pathway that leads to the adhesion of PMNs to endothelial cells.
Highlights
Foam cells beneath an intact arterial endothelial lining [1, 2]
TNF␣ Stimulates LacCer Synthesis in a Time- and Concentration-dependent Fashion in human umbilical vein endothelial cells (HUVECs)—We found that TNF-␣ stimulated GalT-2 activity in a time-dependent manner (Fig. 1A)
This phenomenon was accompanied by a time-dependent increase in the synthesis of LacCer as evidenced by an increase in the incorporation of [3H]galactose into LacCer in cells stimulated with TNF-␣ (Fig. 1B)
Summary
Foam cells beneath an intact arterial endothelial lining [1, 2]. Localized attachment of circulating monocytes and lymphocytes to the arterial endothelium appears to precede the formation of early foam cell lesions [1, 2]. VCAM-1, which is induced on endothelial cells by IL-1, TNF-␣, or IL-4 [9] serves as a ligand for very late antigen-4 on lymphocytes, monocytes, and eosinophils [10]. ICAM-1 is a specific ligand for lymphocyte function-associated antigen-1 [11] and Mac-1 (CD11/CD18), which is expressed on neutrophils and monocytes [12]. In vivo studies have suggested that both ICAM-1 and VCAM-1, which are inducible by TNF-␣, are expressed in human atherosclerotic lesions [13]. It is well documented that TNF-␣ induces the expression of cell adhesion molecules by activating an oxidantsensitive signal transduction pathway [14, 15]. LacCer Mediates TNF-␣ Signaling human umbilical vein endothelial cells (HUVECs) that facilitate the adhesion of neutrophils
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