Thrombin Stimulated Reactive Oxygen Species Production in Cultured Human Endothelial Cells

Abstract

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Pollutant particles enhanced H2O2production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells

“…Other enzyme systems that are potential sources for H 2 O 2 , including xanthine oxidase, cytochrome P-450, NO synthase, and cyclooxygenase-1, were less likely sources because their respective inhibitors, (allupurinol, cimetidine, L-NAME, and indomethacin) did not inhibit UPinduced H 2 O 2 release. The NAD(P)H oxidase activity in vascular endothelial cells can be stimulated by many plasma membranemediated events, including cytokines, hormones, growth factors, G protein receptor agonists, and shear forces, and activation of NAD(P)H oxidase is known to be involved in the pathogenesis of vascular disease (15,20,21,24,34). Electron leaks from the mitochondrial electron transport chain of vascular endothelial cells, on the other hand, have been observed with different stimuli, such as hypoxia-reoxygenation and glucocorticoid excess (28,43).…”

The Pickering Lecture British Hypertension Society, 10th September 2002

“…Stimulation of vascular smooth muscle cells with thrombin, plateletderived growth factor, tumour necrosis factor-α, and lactosylceramide all increase activity of the vascular ROS formation and NAD(P)H oxidase activity. [43][44][45][46] Exposure of human umbilical endothelial cells to 5 or 20 dyne/cm 2 unidirectional laminar shear stress results in a transient elevation in NADH-dependent O 2 G-formation, whereas oscillatory shear caused a sustained increase in oxidase activity. 47 Both the endothelial and vascular smooth muscle oxidases have also been reported to be activated by cyclic stretch.…”

Defects in microvascular haemodynamics during prediabetes: contributor or epiphenomenon?

“…Again here the insulin resistant state itself, rather than hyperinsulinaemia, could be responsible for the production of free radicals through an activation of NADH/NADPH oxidase [37]. Iron, through increased ferritin concentrations [38] and thrombin [39] are favouring factors for oxidative stress. In low birthweight infants, oxidant status was impaired [40].…”