Endothelial NADPH Oxidase: Mechanism of Activation by Low-Density Lipoprotein

Abstract

Exposure to atherogenic levels of low-density lipoprotein (LDL) causes elevated reactive oxygen species (ROS) production by human endothelial cells (ECs). NADPH oxidase is thought to be the main source of ROS generated by LDL-activated ECs. The mechanism by which this lipoprotein activates endothelial NADPH oxidase is incompletely understood. To gain further insight into the signaling pathway, the authors have examined the effects of inhibitors to various signal transducing enzymes, including the G(i)-protein coupled receptor (pertussis toxin), Src tyrosine kinase (PP1), phospholipase C-gamma (U73122), phosphatidylinositol 3-kinase (LY294002), p42/p44 mitogen-activated protein kinase (MAPK) kinase (PD98059), p38 MAPK (SB203580), protein kinase C (Ro 318220, GF 109203X, Go 6976), and cytosolic phospholipase A(2) (AACOCF3), on the ROS-producing capacity ECs activated by LDL. Exposure of cultured ECs to LDL (0.45 mg protein/mL) stimulated ROS formation, as measured using a 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate assay. This effect was partially inhibited by Ro 318220, GF 109203X, U73122, and SB203580, and blocked or nearly completely inhibited by PP1, pertussis toxin, LY294002, PD98059, and AACOCF3. Only a partial, minor inhibition occurred with the protein kinase C inhibitor, Go 6976. These results are most consistent with LDL activating endothelial NADPH oxidase, predominantly through a signaling pathway that leads to cytosolic phospholipase A(2) activation.

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

“…Phosphorylation of cPLA 2 and release of arachidonic acid were demonstrated in vascular smooth muscle cells stimulated with Ang II, although this was not directly linked to NOX2 activation (33). ROS production by endothelial cells activated with low density lipoprotein was inhibited by the presence of the cPLA 2 inhibitor, AACOCF 3 (35). The possible role for cPLA 2 in NOX2 activation in PMN and monocytes has been inferred by its presence on the plasma membrane following stimulation and by decreased ROS production when expression of cPLA 2 was "knocked down" with antisense oligodeoxyribonucleotides (31,36,37).…”

“…Previous studies have provided evidence that PLA 2 activity is important for NOX2 activation in PMN and possibly endothelial and other cell types (30,(33)(34)(35). However, assessment of the specific PLA 2 involved in activation has been hampered by the relative non-specificity of inhibitors used in many of the earlier studies.…”

Vascular NADPH oxidases: molecular mechanisms of activation

“…Accordingly, oxidized LDL increases vascular and endothelial cells radical generation [130] and transfection of p22phox antisense oligonucleotides blocked this effect [131]. The LPC-mediated activation of the endothelial NADPH oxidase is sensitive to inhibitors of phospholipase A2, PKC, and PI3-K [132].…”

TNF-α potentiates protein-tyrosine nitration through activation of NADPH oxidase and eNOS localized in membrane rafts and caveolae of bovine aortic endothelial cells

“…Like neutrophils and other vascular cell types, including smooth muscle and cardiac myocytes (2,25), endothelial cells express the major functional subunits of NADPH oxidase (4,30,35). The importance of this enzyme system has been highlighted in recent studies showing that lipoproteins (45), cytokines (10,18,36), endothelin (13), ANG II (34,38,64), and oscillatory shear stress (43,55) can induce NADPH oxidase-dependent release of ROS. In addition, both the proliferative and migratory effects of vascular endothelial growth factor was shown to depend on ROS generated by NADPH oxidase (1).…”