Lactosylceramide Mediates Shear-Induced Endothelial Superoxide Production and Intercellular Adhesion Molecule-1 Expression

Abstract

Laminar shear stress activates NADPH oxidase in vascular endothelial cells (ECs), and the generated superoxide radicals (O₂^–·) are known to be involved in intercellular adhesion molecule (ICAM)-1 expression. In this study, the role of a glycosphingolipid (GSL), lactosylceramide (LacCer), as a second messenger in the shear-induced O₂^–· generation and ICAM-1 expression was examined. It is known that glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer) from ceramide, and subsequently lactosylceramide synthase (GalT-2) synthesizes LacCer from GlcCer. We observed that exposing cultured human umbilical vein ECs (HUVECs) to fluid shear stress (20 dyn/cm² for 30 min) activated GalT-2. Shear stress also increased EC O₂^–· generation, that peaked at 30 min, and surface ICAM-1 protein expression at 6 h post-shear. EC preincubation with the antioxidant N-acetylcysteine (NAC; 20 mM for 2 h) completely abolished the shear-induced O₂^–· production and significantly inhibited ICAM-1 expression. EC preincubation with D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of the GSL glycosyltransferases GlcT-1 and GalT-2, abrogated the shear-induced activation of GalT-2. D-PDMP also abolished the shear-induced O₂^–· production and ICAM-1 expression. We conclude that laminar shear stress activates GalT-2 to produce LacCer. In turn, LacCer activates NADPH oxidase, which produces O₂^–·, and O₂^–· mediates the shear-induced increase in ICAM-1 expression. Thus, LacCer may play an important role in hemodynamic force-induced pathological conditions, such as atherosclerosis and ischemia/reperfusion injury.

Lactosylceramide Mediates the Expression of Adhesion Molecules in TNF-α and IFNγ-stimulated Primary Cultured Astrocytes

“…Previous studies reported that LacCer mediates a series of processes, such as ROS generation, Ras activation, ERK and JNK activation, AP-1 and NF-κB activation, and ICAM-1 expression in inflammatory or shear stress conditions [3,5,6,9,10,28]. Interestingly, LacCer itself increased expression of ICAM-1 in HUVECs [9].…”

Ceramide: A common pathway for atherosclerosis?

“…Superoxide production was noted to be stimulated 4-fold when aortic smooth muscle cells were incubated with lactosylceramide (LacCer) [14]. Similarly, Yeh et al noted that the only analogue of ceramide to induce superoxide generation was LacCer, via a plasma membrane associated NADPH oxidase [59]. Zhang and colleagues found that ceramide caused a time-dependent rise in superoxide anion relative to controls and that this was attenuated by treatment with NADPH oxidase inhibitor.…”

Recent advances in the immunobiology of ceramide

“…Preincubation of cells with the antioxidant Nacetylcysteine (NAC) was found to completely abolish the shear-induced superoxide production and significantly inhibit the ICAM-1 expression. Also, D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), attenuates the shear-induced activation of GalT-2, superoxide production and ICAM-1 expression (Yeh et al, 2001;Chatterjee, 1998). The triggering of an inflammatory reaction following activation of transcription factors NF-κB and AP-1 causes phenotypic modulation of medial vascular smooth muscle cells and their migration and subsequent proliferation in the intimal layer; increased extracellular matrix production by these vascular smooth muscle cells increases the volume of intimal tissue causing neointimal hyperplasia, ultimately leading to restenosis (Krueger et al, 2006;Mitra and Agrawal, 2006).…”